Pharmaceuticals and methods for treating hypoxia and screening methods therefor

ABSTRACT

Light-generating fusion proteins having a ligand binding site and a light-generating polypeptide moiety and their use as diagnostics, in drug screening and discovery, and as therapeutics, are disclosed. The light-generating fusion protein has a feature where the bioluminescence of the polypeptide moiety changes upon binding of a ligand at the ligand binding site. The ligand may be, for example, an enzyme present in an environment only under certain conditions, e.g., ubiquitin ligase in a hypoxic state, such that the light-generating fusion protein is “turned on” only under such conditions.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/433,992, filed Jun. 6, 2019, which is a continuation of U.S.application Ser. No. 15/594,454, filed on May 12, 2017, now U.S. Pat.No. 10,365,278, which is a continuation of U.S. application Ser. No.14/328,588, filed on Jul. 10, 2014, now U.S. Pat. No. 9,766,240; whichis a continuation of U.S. application Ser. No. 13/066,033, filed on Apr.4, 2011, now U.S. Pat. No. 8,809,011; which is a continuation of U.S.application Ser. No. 11/879,300, filed on Jul. 17, 2007, now U.S. Pat.No. 7,985,563; which is a continuation of U.S. application Ser. No.11/027,273, filed on Dec. 30, 2004, now abandoned; which is acontinuation of U.S. application Ser. No. 10/859,935, filed Jun. 2,2004, now abandoned; which is a continuation of U.S. application Ser.No. 10/101,812, filed Mar. 19, 2002, now U.S. Pat. No. 6,855,510; whichclaims priority to U.S. application Ser. Nos. 60/277,425, filed Mar. 20,2001; 60/277,431, filed Mar. 20, 2001; 60/277,440, filed Mar. 20, 2001;60/332,493, filed Nov. 9, 2001; 60/332,334, filed Nov. 9, 2001;60/345,200, filed Nov. 9, 2001; 60/345,131 filed Dec. 20, 2001,60/342,598, filed Dec. 20, 2001; and 60/345,132, filed Dec. 20, 2001each of which are incorporated herein by reference in their entireties.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numberCA076120 awarded by The National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

The invention relates to drug discovery. The invention features the useof light emitting proteins as tools for diagnosis, drug screening anddiscovery, and as pharmaceuticals for in vivo treatment.

A key advance in the biomedical arts has been the discovery ofbioluminescent protein moieties, e.g., green fluorescent protein (GFP)and luciferase, which can be expressed in diverse mammalian cell typesand thus act as detectable signals for biological signal transductionpathways and events. An increased understanding of how diversebiological processes are regulated by the actions of cellular enzymes,e.g., kinases, proteases, and ubiquitin ligases, has also been emerging.Alterations in the activity of these enzymes may underlie the initiationand/or progression of diseases such as cancer.

Methods of detecting biological activities and substances usingbioluminescent proteins have recently been developed. For example,protein phosphorylation events can be detected using fusion proteinscontaining GFP (see, e.g., U.S. Pat. No. 5,958,713) or luciferase,aequorin and obelin (see, e.g., U.S. Pat. No. 5,683,888).Light-generating moieties have been introduced into mammals tospecifically localize events such as parasite infection (see, e.g., U.S.Pat. No. 5,650,135).

How cells sense changes in ambient oxygen is a central problem inbiology. In mammalian cells, lack of oxygen, or hypoxia, leads to thestabilization of a sequence-specific DNA-binding transcription factorcalled HIF (hypoxia-inducible factor), which transcriptionally activatesa variety of genes linked to processes such as angiogenesis and glucosemetabolism.

Tissue ischemia is a major cause of morbidity and mortality. Ischemiacan result from chronic hypoxia brought on by lack of blood supply tothe tissue occurring from, for example, stroke, deep vein thrombosis,pulmonary embolus, and renal failure. Ischemic tissue is also found intumors.

HIF binds to DNA as a heterodimer consisting of an alpha subunit and abeta subunit (also called aryl hydrocarbon receptor nuclear translocatoror ARNT). The alpha subunit is rapidly polyubiquitinated and degraded inthe presence of oxygen whereas the beta subunit is constitutivelystable. von Hippel-Lindau (VHL) disease is a hereditary cancer syndromecharacterized by the development of highly vascular tumors thatoverproduce hypoxia-inducible mRNAs such as vascular endothelial growthfactor (VEGF). The product of the VHL tumor suppressor gene, pVHL, is acomponent of multiprotein complex that contains elongin B, elongin C,Cu12, and Rbx1. This complex bears structural and functional similarityto SCF (Skp1/Cdc53 or CullinIF-box) ubiquitin ligases. In the presenceof oxygen pVHL binds directly to HIFα subunits and targets them forpolyubiquitination and destruction. Cells lacking functional pVHL cannotdegrade HIF and thus overproduce mRNAs encoded by HIF target genes.

Progress has also been made in recent years towards understanding themolecular mechanisms in control of cell proliferation. Aberrant cellproliferation is a hallmark event in the onset and progression ofdiseases such as cancer. Progression through the mammalian cell-cycle islinked to the orchestrated appearance and destruction of cyclins.Different cyclins are associated with different cell-cycle transitions.For example, cyclin E is active in late G1 and early S-phase, cyclin Ais active in S-phase, and cyclin B is active in mitosis. Cyclins bind tocyclin-dependent kinases (cdks). In this context, cyclins activate thecatalytic activity of their partner cdk(s) and also play roles insubstrate recognition.

Some transcriptional regulatory proteins, such as the pRB homologs p107and p130, the E2F family members E2F1, E2F2, and E2F3, thetranscriptional coactivator p300, and NPAT (nuclear protein mapped tothe AT locus) form stable complexes with cyclin A/cdk2 and/or cyclinE/cdk2. All of these proteins bind directly or indirectly to DNA. Thus,such complexes might serve as vehicles for increasing the concentrationof cyclin A/cdk2 or cyclin E/cdk2 at certain sites within the genome. Assuch, cyclin A/cdk2 and cyclin E/cdk2 might play relatively direct rolesin processes such as transcription and DNA replication. These twoprocesses are fundamental in normal cell proliferation and are perturbedduring aberrant cell proliferation, such as in cancer.

SUMMARY OF THE INVENTION

The invention relates in part to the discovery of light-generatingfusion proteins (or a cell expressing the light-generating fusionprotein), wherein the light-generating fusion protein features a ligandbinding site and a light-generating polypeptide moiety. Thelight-generating fusion protein (“LGP”) has a feature where the lightgeneration of the light-generating polypeptide moiety changes uponbinding of a ligand at the ligand binding site. The ligand may be activein an environment only under certain conditions, e.g., in a hypoxicstate, such that the light-generating fusion protein is “turned” on oroff only under such conditions.

The light-generating fusion proteins of the invention may be used forscreening for modulators of activity or latency of (or predispositionto) disorders such as hypoxia, cancer, diabetes, heart disease orstroke. For example, a test compound may be administered to a testanimal at increased risk for such a disorder, wherein the test animalrecombinantly expresses a light-generating fusion protein, allowing forlocalization of the light-generating fusion protein, detecting theluminescence of the light-generating polypeptide moiety in the testanimal after administering the test compound, and comparing theluminescence of the light-generating polypeptide moiety in the testanimal with the luminescence of the light-generating polypeptide moietyin a control animal not administered the test compound. A change in theactivity of the light-generating polypeptide moiety in the test animalrelative to the control animal indicates the test compound may be amodulator of, e.g., prolyl hydroxylase.

The effects of an anti-hypoxic compound in vivo may be determined inanother embodiment, by administering to a subject, e.g., mammalian, alight-generating fusion protein comprising an ubiquitin ligase bindingsite and a light-generating polypeptide moiety or a cell expressing thelight-generating fusion protein, allowing for localization of thelight-generating fusion protein or cell in hypoxic tissue in thesubject; and measuring the luminescence of the localizedlight-generating fusion protein from the hypoxic tissue.

Methods in accordance with the invention are also provided fordetermining the effects of an anti-cell proliferation compound understudy in a mammalian subject, by administering to a test subject alight-generating fusion protein containing a cyclin/cdk binding site ora cell expressing the light-generating fusion protein of the invention,allowing for localization of the fusion protein or cell, measuringluminescence from the localized light-generating fusion protein; andimaging the localized light-generating fusion protein, therebydetermining the effects of the anti-cell proliferation compound.

Cyclin/cdk activity may be assayed in another embodiment of theinvention wherein a test sample is contacted with a light-generatingfusion protein comprising a cyclin/cdk binding site and alight-generating polypeptide moiety; and thereafter the presence oramount of cyclin/cdk activity in said test sample is determined bymeasuring the luminescence of the test sample.

The invention yet further relates to DNA constructs, including anisolated DNA encoding a modified LGP wherein one or more amino acidshave been substituted, inserted or deleted to provide a ligand bindingsite such as a ubiquitin ligase or protease recognition site, whereinfluorescence of the LGP changes upon binding of a ligand to the ligandbinding site.

The invention also relates to light-generating fusion proteins having aligand binding site, such as a ubiquitin ligase binding site, or a HIF1αpolypeptide moiety; and a light generating polypeptide moiety. Anotherembodiment features a light-generating fusion protein with a cyclin/cdkbinding site, a suicide protein polypeptide moiety, and alight-generating polypeptide moiety; and a light-generating fusionprotein comprising a protein dimerization domain and a light-generatingprotein moiety. In a preferred embodiment, the light-generating fusionproteins have the property that upon ligand binding to the ligandbinding site, the luminescence of the light-generating polypeptidemoiety is changed without altering the phosphorylational state of thefusion protein.

The invention further relates to fusion proteins including a HIF1αpolypeptide moiety or cyclin/cdk binding site, and a suicide proteinpolypeptide moiety. A light-generating polypeptide moiety may optionallybe included. These protein constructs may be used to selectively targetcertain cells, e.g., hypoxic tumor cells, for destruction. For example,the invention includes methods of treating hypoxic or ischemic disordersby administering to a subject an effective amount of a fusion proteincomprising a HIFα polypeptide moiety having a binding affinity forprolyl hydroxylase, and a suicide polypeptide moiety, such that thehypoxic or ischemic disorder is treated. Methods of killing hypoxictumor cells, wherein an effective amount of the fusion/suicide proteinis administered to a subject, such that the hypoxic tumor cells arekilled; and methods of treating cell-proliferating disorders byadministering to a subject an effective amount of a fusion proteincomprising a cyclin/cdk binding site and a suicide protein polypeptidemoiety, such that the cell-proliferating disorder is treated, are alsocontemplated.

The treatment of cell-proliferating disorders may be monitored by anembodiment of the invention, e.g., by administering to a subject aneffective amount of a fusion protein comprising a HIFα polypeptidemoiety having a binding affinity for prolyl hydroxylase, a suicidepolypeptide moiety, and a light-generating polypeptide moiety, whereinthe light generation of the light-generating fusion protein changes uponbinding of prolyl hydroxylase to the HIFα polypeptide moiety, such thatthe cell-proliferating disorder is treated, and monitoring the abilityof the fusion protein to inhibit cell proliferation by measuring thelight generated by the light-generating fusion protein. Alternately,treatment of cell-proliferating disorders may be monitored byadministering to a subject an effective amount of a light-generatingfusion protein comprising a cyclin/cdk binding site, a suicide proteinpolypeptide moiety, and a light-generating polypeptide moiety, whereinthe light generation of the light generating fusion protein changes uponbinding of a cyclin to the cyclin/cdk binding site, such that saidcell-proliferating disorder is treated; and monitoring the ability ofthe fusion protein to inhibit cell proliferation by measuring the lightgenerated by the light-generating fusion protein.

Other embodiments of the invention include a cyclin/cdk binding site anda light generating polypeptide moiety; and a protease binding site and alight-generating protein moiety. Antibodies specific for a proteincomplex comprising HIF1α and pVHL are also detailed as within thepresent invention.

BRIEF DESCRIPTION OF THE DRAWING

FIGS. 1A, 1B, 1C, and 1D are schematic representations of differentfusion proteins of the present invention.

FIGS. 2A and 2B are second schematic representations of different fusionproteins of the invention.

FIGS. 3A, 3B, 3C, and 3D shows pVHL binding to a modified form of HIF.

FIGS. 4A, 4B, 4C, and 4D shows pVHL binding to a HIF1α-derived peptideif Leu562 and Pro564 are intact.

FIGS. 5A, 5B, and 5C shows ubiquitination and degradation of HIF linkedto Leu562 and Pro 564.

FIGS. 6A, 6B, 6C, and 6D depicts proline hydroxylation linked topVHL-binding.

FIGS. 7A, 7B, 7C, and 7D illustrates pVHL specifically recognizing HIF1αwith hydroxylated proline 564.

FIGS. 8A, 8B, and 8C illustrates the production of TETr-cyclins A and E.

FIGS. 9A, 9B, and 9C shows DNA bound cyclins A and E differentiallyaffecting transcription.

FIGS. 10A, 10B, and 10C illustrates transcriptional regulation bycyclins A and E dependent upon DNA binding.

FIGS. 11A, 11B, and 11C depicts cyclin box is required fortranscriptional repression by DNA bound cyclin A.

FIGS. 12A, 12B, and 12C illustrates that transcriptional activation bycyclin E is linked to its ability to bind to cdk2 and interact withsubstrates.

FIGS. 13A, 13B, and 13C shows that transcriptional activation by DNAbound cyclin E depends on cdk2 catalytic activity.

FIGS. 14A-1, 14A-2, 14A-3, 14A-4, 14A-5, 14A-6, 14A-7, 14B, and 14Cshows transcriptional effects mediated by cell-cycle dependent changesin endogenous cyclins E and A.

FIGS. 15-18 illustrate the results obtained in Example 2.

FIG. 19 illustrates the wild type sequence of HIF1α, Accession No.Q16665 (SEQ ID NO:23).

FIG. 20 is a schematic representation of a nucleic acid encoding alight-generating fusion protein of the invention.

DETAILED DESCRIPTION OF THE INVENTION Definitions

“Light-generating” or “luminescent” includes the property of generatinglight through a chemical reaction or through the absorption ofradiation, including phosphorescence, fluorescence, and bioluminescence.

“Light” includes electromagnetic radiation having a wavelength ofbetween about 300 nm and about 1100 nm.

“Non-invasive” methods for detecting localization in a subject does notinclude largely invasive methods such as conventional surgery or biopsy.

“Light-generating fusion protein” includes proteins of the inventionhaving a light generating or luminescent portion, i.e., alight-generating polypeptide moiety and a ligand binding site. Ingeneral, when a ligand of interest binds to the ligand binding site ofthe light-generating fusion protein, the light-generating properties ofthe light-generating polypeptide moiety change, either going from “dark”to “light”, or vice versa.

“Light-generating polypeptide moiety” includes any protein known tothose of ordinary skill in the art to provide a readily detectablesource of light when present in stable form. Non-limiting examplesinclude light-generating proteins described in U.S. Pat. Nos. 5,683,888,5,958,713, and 5,650,135, e.g., ferredoxin IV, green fluorescentprotein, red fluorescent protein, yellow fluorescent protein, bluefluorescent protein, the luciferase family and the aequorin family. In apreferred embodiment, the light-generating polypeptide moiety is aprotein such as green fluorescent protein, red fluorescent protein,yellow fluorescent protein and blue fluorescent protein.

“Colinear effector site” includes regions of the light-generatingpolypeptide moiety that, when acted on by events subsequent to ligandbinding, cause the light-generating polypeptide moiety to change itspresent light-emitting state (i.e., on or off). These regions making upthe colinear effector site may do this by, e.g., conformationaldistortion, chemical modification, e.g., ubiquitination of a residue orresidues in the colinear effector site, or by cleavage of a portion ofall or part of the colinear effector site.

“Having binding character for prolyl hydroxylase” refers to a propertymaking HIFα polypeptide moieties suitable for, e.g., screening methodsof the invention and includes HIF polypeptide sequences suitable oradapted for prolyl hydroxylase binding as well as native or wild-typeHIF sequences to which pVHL has recognized and bound.

“Having binding character for ubiquitin ligase” refers to a propertymaking HIFα polypeptide moieties suitable for, e.g., screening methodsof the invention, e.g., including native HIF polypeptide sequenceshaving hydroxylated proline residues hydroxylated by prolyl hydroxylase,or, e.g., “HIF1α polypeptide moieties” as defined herein.

“Localization” includes determining the particular region of the subjectof an entity of interest, e.g., a tumor.

“Kemptide” includes a synthetic cAMP peptide substrate corresponding topart of the phosphyorylation site sequence in porcine liver pyruvatekinase. “Malantide” includes cAMP-dependent protein kinase and proteinkinase C substrate in various tissues.

“Small molecule” includes compositions that have a molecular weight ofless than about 5 kD and most preferably less than about 4 kD. Smallmolecules can be, e.g., nucleic acids, peptides, polypeptides,peptidomimetics, carbohydrates, lipids or other organic or inorganicmolecules. “Spread of infection” includes the spreading and colonizationby a pathogen of host sites other than the initial infection site. Theterm can also include, however, growth in size and/or number of thepathogen at the initial infection site.

“Ligand” includes a molecule, a small molecule, a biomolecule, a drug, apeptide, a polypeptide, a protein, a protein complex, an antibody, anucleic acid, or a cell.

“Ligand binding site” includes the location on the light-generatingfusion protein to which a ligand binds, whereupon the light-generatingpolypeptide moiety is activated or inactivated as a direct or indirectconsequence of ligand binding Binding to the ligand binding site may bedirect or indirect, e.g., via protein dimerization in conjunction withother proteins, as described hereinbelow.

“Targeting moiety” includes moieties which allow the light-generatingfusion protein of the invention to be selectively delivered to a targetorgan or organs. For example, if delivery of a therapeutic compound tothe brain is desired, the carrier molecule may include a moiety capableof targeting the compound to the brain, by either active or passivetransport. Illustratively, the carrier molecule may include a redoxmoiety, as described in, for example, U.S. Pat. Nos. 4,540,564 and5,389,623, both to Bodor. These patents disclose drugs linked todihydropyridine moieties which can enter the brain, where they areoxidized to a charged pyridinium species which is trapped in the brain.Thus, compound accumulates in the brain. Many targeting moieties areknown, and include, for example, asialoglycoproteins (see, e.g. Wu, U.S.Pat. No. 5,166,320) and other ligands which are transported into cellsvia receptor-mediated endocytosis. Targeting moieties may be covalentlyor non-covalently bound to a light-generating fusion protein. Thetargeting moiety may also be attached to a vector.

“Bioluminescent” molecules or moieties include luminescent substanceswhich utilize chemical energy to produce light.

“Fluorescent” molecules or moieties include those which are luminescentvia a single electronically excited state, which is of very shortduration after removal of the source of radiation. The wavelength of theemitted fluorescence light is longer than that of the excitingillumination (Stokes' Law), because part of the exciting light isconverted into heat by the fluorescent molecule.

“Entities” include, without limitation, small molecules such as cyclicorganic molecules; macromolecules such as proteins; polymers; proteins;polysaccharides; nucleic acids; particles, inert materials; organelles;microorganisms such as viruses, bacteria, yeast and fungi; cells, e.g.,eukaryotic cells; embryos; prions; tumors; all types of pathogens andpathogenic substances; and particles such as beads and liposomes. Inanother aspect, entities may be all or some of the cells that constitutethe mammalian subject being imaged, e.g., diseased or damaged tissue, orcompounds or molecules produced by those cells, or by a condition understudy. Entities for which the invention has particular utility includetumors, proliferating cells, pathogens, and cellular environmentscomprising hypoxic tissue.

“Infectious agents” include parasites, viruses, fungi, bacteria orprions.

“Promoter induction event” includes an event that results in the director indirect induction of a selected inducible promoter.

“Heterologous gene” includes a gene which has been transfected into ahost organism. Typically, a heterologous gene refers to a gene that isnot originally derived from the transfected or transformed cells'genomic DNA.

“Opaque medium” includes a medium that is “traditionally” opaque, notnecessarily absolutely opaque. Accordingly, an opaque medium includes amedium that is commonly considered to be neither transparent nortranslucent, and includes items such as a wood board, and flesh and skinof a mammal.

“HIFα polypeptide moiety” includes all or part of the amino acidsequence of HIF1α, HIF2α, or HIF3α.

“HIF1α polypeptide moiety” includes all or part of the amino acidsequence of HIF1α, e.g., SEQ ID NO: 23, the amino acid sequencecorresponding to the N-terminal residues 1-600 of HIF1α, numbered inaccordance with wild-type HIF1α, wherein either or both of residues 402and 564 are proline or hydroxylated proline, or an 80 to 120, 20 to 30,12 to 14, or 4 to 12 amino acid sequence corresponding to the residuesadjacent to and/or surrounding residue 402 and/or 564, inclusive, ofHIF1α, wherein residues 402 and/or 564 is proline or hydroxylatedproline.

The invention relates in part to methods and compositions relating todetecting, localizing and quantifying enzyme activities andprotein-protein interactions in vivo, in vitro and in silico usinglight-emitting fusion proteins. The fusion proteins contain domainscapable of binding by enzymes and other ligands, and of being modifiedas a consequence of this binding. The light generating domains include,without limitation, regions from fluorescent proteins and bioluminescentproteins. Light emission is detected by known methods, such as detectionwith suitable instrumentation (such as a CCD camera) in vivo, in vitroor in silico, such as in a living cell or intact organism, a cellculture system, a tissue section, or an array.

Light-generating fusion proteins of the invention are capable of takingpart in a luminescent reaction whereby different biological,biochemical, chemical and physical events are measured. Thelight-generating fusion protein is capable of being modified such thatit does or does not emit light or cause light to be emitted.Light-generating fusion proteins include a ligand binding site and alight-generating polypeptide moiety, wherein the bioluminescence of thepolypeptide moiety changes upon binding of a ligand at the ligandbinding site.

Without wishing to be bound by interpretation, the ligand binding siteacts as in a sense as a “switch” for the light-generating polypeptidemoiety, i.e., when the ligand binds to the ligand binding site, thelight-generating polypeptide moiety emits light, or alternately, ceasesto do so upon ligand binding. The “switching” on or off, in embodiment,may be done by means of a “colinear effector site” which includesregions of the light-generating polypeptide moiety that, when acted onby events subsequent to ligand binding, cause the light-generatingpolypeptide moiety to change its present light-emitting state (i.e., onor off). The regions making up the colinear effector site may do thisby, e.g., conformational distortion, chemical modification, e.g.,ubiquitination of a residue or residues in the colinear effector site,or by cleavage of a portion of all or part of the colinear effectorsite.

The invention further provides methods for testing putative inhibitorcompounds for activity (“screening”) in promoting HIF stabilization,e.g.; contacting the compound, ischemic tissue, and the fusion proteinof the invention under conditions appropriate to detect the fusionprotein if the compound promotes HIF stabilization. The method (alsoreferred to herein as a “screening assay”) can be used for identifyingmodulators, i.e., candidate or test compounds or agents (e.g., peptides,peptidomimetics, small molecules or other drugs) that promote HIFstabilization. The invention also includes compounds identified in thescreening assays described herein, and pharmaceutical compositions fortreatments as described herein.

Other screening methods are also part of the invention. For example,modulators of activity or latency of, or predisposition to disorders maybe identified by administering a test compound to a test animal atincreased risk for a disorder (e.g., cancer, diabetes, heart disease,stroke, or a hypoxia-related disorder), wherein the test animalrecombinantly expresses a light generating fusion protein comprising aligand binding site and a light-generating polypeptide moiety, whereinthe light generation of the light-generating fusion protein changes uponbinding of a ligand at the ligand binding site, and the ligand bindingsite recognizes a ligand on an entity associated with a disorder, or aproduct of the disorder; allowing for localization of thelight-generating fusion protein and an entity, wherein contact betweenthe ligand binding site and a ligand associated with the disorder causesa modification of a colinear effector site which alters the lightgeneration of the light-generating polypeptide moiety; detecting theluminescence of the light-generating polypeptide moiety in the testanimal after administering the compound; and comparing the luminescenceof the light-generating polypeptide moiety in the test animal with theluminescence of the light-generating polypeptide moiety in a controlanimal, wherein a change in the activity of the light-generatingpolypeptide moiety in the test animal relative to the control indicatesthe test compound is a modulator of latency of or predisposition to, thedisorder in question.

The invention advantageously may be used to non-invasively determine theeffects of an anti-hypoxic compound in vivo. A light-generating fusionprotein of the invention, or a cell expressing same, comprising anubiquitin ligase binding site and a light-generating polypeptide moiety,wherein the light generation of the light-generating fusion proteinchanges upon binding of a ubiquitin ligase at the ubiquitin ligasebinding site, the ubiquitin ligase binding site recognizing a ubiquitinligase present in hypoxic conditions in hypoxic tissue is administeredto a subject. Localization of the light-generating fusion protein orcell in hypoxic tissue in the subject (wherein contact between theubiquitin ligase binding site and a ubiquitin ligase causes amodification of a colinear effector site which alters the lightgeneration of the light-generating polypeptide moiety) is allowed tooccur, and the ability of the candidate compound to inhibit hypoxia isdetermined, by measuring the luminescence of the localizedlight-generating fusion protein.

The invention further relates to methods of identifying or detectingprolyl hydroxylation, wherein the substrate peptide (or polypeptide) iscontacted with pVHL, wherein the amount of pVHL bound reflects thedegree of hydroxylation. In one embodiment, the peptide corresponds toHIF1a 555-575. The HIF peptide can be immobilized (for example, on anitrocellulose filter or at the bottom of a 96 well plate) or free insolution. Binding to pVHL can be monitored using a variety of standardmethods familiar to those skilled in the art.

In another embodiment, the invention relates to methods of identifyingor detecting prolyl hydroxylation, wherein the substrate peptide orpolypeptide is contacted with an antibody, wherein the amount ofantibody bound reflects the degree of hydroxylation. In one embodiment,the peptide corresponds to HIF1α 555-575. The HIF peptide may beimmobilized (for example, on a nitrocellulose filter or at the bottom ofa 96 well plate) or free in solution. Binding to the antibody can bemonitored using a variety of standard methods familiar to those skilledin the art.

Yet another embodiment of the invention relates to methods ofidentifying or detecting prolyl hydroxylation wherein a polypeptide istranslated in the presence of labeled, e.g., radioactive, proline and aprolyl hydroxylase, hydrolyzing the resulting labeled polypeptide, anddetecting labeled hydroxyproline incorporation by analytical means, suchas thin layer chromatography.

A further embodiment of the invention relates to methods of identifyingor detecting prolyl hydroxylation wherein the substrate peptide (orpolypeptide) is contacted with a source of prolyl hydroxylase in thepresence or absence of putative inhibitors, and the degree of prolylhydroxylation is monitored as described in anyone of the above threeparagraphs. In one embodiment, the peptide corresponds to HIF1α 555-575,and the prolyl hydroxylase consists of a mammalian cell extract. Inanother embodiment, the peptide corresponds to HIF1α 555-575 and theprolyl hydroxylase consists of purified or partially purified Eg19.Further, particularly useful embodiments relate to small moleculeinhibitors of prolyl hydroxylation such as identified using this method,and use of the inhibitors to treat diseases characterized by ischemia.

The test compounds of the invention can be obtained using any of thenumerous approaches in combinatorial library methods known in the art,including: biological libraries; spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; the “one-bead one-compound” library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary approach is limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds. See, e.g., Lam, 1997. Anticancer DrugDesign 12: 145.

Libraries of chemical and/or biological mixtures, such as fungal,bacterial, or algal extracts, are known in the art and can be screenedwith any of the assays of the invention. Examples of methods for thesynthesis of molecular libraries can be found in the art, for examplein: DeWitt, et al., 1993. Proc. Natl. Acad. Sci. U.S.A. 90: 6909; Erb,et al., 1994. Proc. Natl. Acad. Sci. U.S.A. 91: 11422; Zuckermann, etal., 1994. J. Med. Chem. 37: 2678; Cho, et al., 1993. Science 261: 1303;Carrell, et al., 1994. Angew. Chem. Int. Ed. Engl. 33:2059; Carell, etal., 1994. Angew. Chem. Int. Ed. Engl. 33: 2061; and Gallop, et al.,1994. J. Med. Chem. 37: 1233.

Libraries of compounds may be presented in solution (e.g., Houghten,1992. Biotechniques 13: 412-421), or on beads (Lam, 1991. Nature 354:82-84), on chips (Fodor, 1993. Nature 364: 555-556), bacteria (Ladner,U.S. Pat. No. 5,223,409), spores (Ladner, U.S. Pat. No. 5,233,409),plasmids (Cull, et al., 1992. Proc. Natl. Acad. Sci. USA 89: 1865-1869)or on phage (Scott and Smith, 1990. Science 249: 386-390; Devlin, 1990.Science 249:404-406; Cwirla, et al., 1990. Proc. Natl. Acad. Sci. U.S.A.87: 6378-6382; Felici, 1991. J. Mol. Biol. 222: 301-310; Ladner, U.S.Pat. No. 5,233,409.).

Modulation of prolyl hydroxylase has a variety of uses. Inhibitingprolyl hydroxylase may facilitate cell cycle progression and theproduction of a number of proteins which promote angiogenesis and/orpromote cellular survival or cellular function in hypoxia, a desirableoutcome in the treatment of certain clinical conditions, particularlyischemic conditions such as coronary, cerebral and vascularinsufficiency.

VHL used in assays of the invention may be any suitable mammalian VHL,particularly human VHL. Human VHL has been cloned and sources of thegene can be readily identified by those of ordinary skill in the art.Its sequence is available as Genbank accession numbers AF010238 andL15409. Other mammalian VHLs are also available, such as murine VHL(accession number U12570) and rat (accession numbers U14746 and S80345).Non-mammalian homologues include the VHL-like protein of C. elegans,accession number F08G12. VHL gene sequences may also be obtained byroutine cloning techniques, for example by using all or part of thehuman VHL gene sequence as a probe to recover and to determine thesequence of the VHL gene in other species. A wide variety of techniquesare available for this, for example, PCR amplification and cloning ofthe gene using a suitable source of mRNA (e.g., from an embryo or aliver cell), obtaining a cDNA library from a mammalian, vertebrate,invertebrate or fungal source, e.g., a cDNA library from one of theabove-mentioned sources, probing the library with a polynucleotide ofthe invention under stringent conditions, and recovering a cDNA encodingall or part of the VHL protein of that mammal. Suitable stringentconditions include hybridization on a solid support (filter) overnightincubation at 420° C. in a solution containing 50% formamide, 5×SSC (750mM NaC1, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6),5×Denhardt's solution, 10% dextran sulfate and 20 μg/m1 salmon spermDNA, followed by washing in 0.03 M sodium chloride and 0.03 M sodiumcitrate (i.e. 0.2×SSC) at from about 50° C. to about 60° C.). Where apartial cDNA is obtained, the full length coding sequence may bedetermined by primer extension techniques.

It is not necessary to use the entire VHL protein (including theirmutants and other variants). Fragments of the VHL may be used, providedsuch fragments retain the ability to interact with the target domain ofthe HIFα subunit. Fragments of VHL may be generated in any suitable wayknown to those of skill in the art. Suitable ways include, but are notlimited to, recombinant expression of a fragment of the DNA encoding theVHL. Such fragments may be generated by taking DNA encoding the VHL,identifying suitable restriction enzyme recognition sites either side ofthe portion to be expressed, and cutting out that portion from the DNA.The portion may then be operably linked to a suitable promoter in astandard commercially available expression system. Another recombinantapproach is to amplify the relevant portion of the DNA with suitable PCRprimers. Small fragments of the VHL (up to about 20 or 30 amino acids)may also be generated using peptide synthesis methods which are wellknown in the art. Generally fragments will be at least 40, preferably atleast 50, 60, 70, 80 or 100 amino acids in size.

The HIFα subunit protein may be any human or other mammalian protein, orfragment thereof which has the ability to bind to a wild type fulllength VHL protein, such that the binding is able, in a normoxiccellular environment, to target the a subunit for destruction.

A number of HIFα subunit proteins have been cloned. These include HIF1α,the sequence of which is available as Genbank accession number U22431,HIF2α, available as Genbank accession number U81984 and HIF3α, availableas Genbank accession numbers AC007193 and AC079154. These are all humanHIFα subunit proteins. HIFα subunit proteins from other species,including murine HIF1α (accession numbers AF003695, US9496 and X95580),rat HIF1α (accession number Y09507), murine HIF2α (accession numbersU81983 and D89787) and murine HIF3α (accession number AF060194). Othermammalian, vertebrate, invertebrate or fungal homologues may be obtainedby techniques similar to those described above for obtaining VHLhomologues.

Variants of the HIFα subunits may be used, such as synthetic variantswhich have at least 45% amino acid identity to a naturally occurringHIFα subunit (particularly a human HIFα subunit), preferably at least50%, 60%, 70%, 80%, 90%, 95% or 98% identity.

Fragments of the HIFα subunit protein and its variants may be used,provided that the fragments retain the ability to interact with awild-type VHL, preferably wild-type human VHL. Such fragments aredesirably at least 20, preferably at least 40, 50, 75, 100, 200, 250 or400 amino acids in size. Alternately, such fragments may be 12 to 14amino acids in size, or as small as four amino acids. Most desirablysuch fragments include the region 555-575 found in human HIF1α or itsequivalent regions in other HIFα subunit proteins. Optionally thefragments also include one or more domains of the protein responsiblefor transactivation. Reference herein to a HIFα subunit protein includesthe above mentioned mutants and fragments which are functionally able tobind VHL protein unless the context is explicitly to the contrary.

The percentage homology (also referred to as identity) of DNA and aminoacid sequences can be calculated using commercially availablealgorithms. The following programs (provided by the National Center forBiotechnology Information) may be used to determine homologies: BLAST,gapped BLAST and PSI-BLAST, which may be used with default parameters.The algorithm GAP (Genetics Computer Group, Madison, Wis.) uses theNeedleman and Wunsch algorithm to align two complete sequences thatmaximizes the number of matches and minimizes the number of gaps.Generally, the default parameters are used, with a gap creationpenalty=12 and gap extension penalty=4. Use of either of the terms“homology” and “homologous” herein does not imply any necessaryevolutionary relationship between compared sequences, in keeping forexample with standard use of terms such as “homologous recombination”which merely requires that two nucleotide sequences are sufficientlysimilar to recombine under the appropriate conditions.

The precise format of the screening assays may be varied using routineskill and knowledge. The amount of VHL, HIFα subunit and, whererequired, further components, may be varied depending upon the scale ofthe assay. In general, the person of skill in the art will selectrelatively equimolar amounts of the two components, say from 1:10 to100:1, preferably from 1:1 to 10:1 molar ratio of VHL to HIFα subunit.However there may be particular assay formats which can be practicedoutside this range.

Where assays of the invention are performed within cells, the cells maybe treated to provide or enhance a normoxic environment. By “normoxic”it is meant levels of oxygen similar to those found in normal air, e.g.about 21% O₂ and 5% CO₂, the balance being nitrogen. Of course, theseexact proportions do not have to be used, and may be variedindependently of each other. Generally a range of from 10-30% oxygen,1-10% CO₂ and a balance of nitrogen or other relatively inert andnon-toxic gas may be used. Normoxia may be induced or enhanced in cells,for example by culturing the cells in the presence of hydrogen peroxideas described above.

Alternatively, or by way of controls, cells may also be cultured underhypoxic conditions. By “hypoxic” it is meant an environment with reducedlevels of oxygen. Most preferably oxygen levels in cell culture will be0.1 to 1.0% for the provision of a hypoxic state. Hypoxia may be inducedin cells simply by culturing the cells in the presence of lowered oxygenlevels. The cells may also be treated with compounds which mimic hypoxiaand cause up regulation of HIFα subunit expression. Such compoundsinclude iron chelators, cobalt (II), nickel (II) or manganese (II), allof which may be used at a concentration of 20 to 500 μM, such as 100 μM.Iron chelators include desferrioxamine, O-phenanthroline orhydroxypyridinones (e.g. 1,2-diethyl hydroxypyridinone (CP94) or1,2-dimethyl hydroxypyridinone (CP20).

Cells in which assays of the invention may be preformed includeeukaryotic cells, such as yeast, insect, mammalian, primate and humancells. Mammalian cells may be primary cells or transformed cells,including tumor cell lines. The cells may be modified to express or notto express other proteins which are known to interact with HIF (xsubunit proteins and VHL protein, for example Elongin C and Elongin Bproteins in the case of VHL and ARNT protein, in the case of HIFαsubunit protein.)

In cell free systems such additional proteins may be included, forexample by being provided by expression from suitable recombinantexpression vectors.

In assays performed in cells, it will be desirable to achieve sufficientexpression of VHL to recruit sufficient HIFα subunit to a complex suchthat the effect of a putative modulator compound may be measured. Thelevel of expression of VHL and HIFα subunit may be varied within fairlywide limits, so that the intracellular levels of the two may vary by awide ratio, for example from 1:10 to 1000:1, preferably 1:1 to 100:1,molar ratio of VHL to HIFα subunit.

The amount of putative modulator compound which may be added to an assayof the invention will normally be determined by trial and errordepending upon the type of compound used. Typically, from about 0.01 to100 μM concentrations of putative modulator compound may be used, forexample from 0.1 to 10 μM. Modulator compounds may be those which eitheragonize or antagonize the interaction. Antagonists (inhibitors) of theinteraction are particularly desirable.

Modulator compounds which may be used may be natural or syntheticchemical compounds used in drug screening programs. Extracts of plantswhich contain several characterized or uncharacterized components mayalso be used.

The invention provides methods for determining hypoxic conditions,cancer or infection in an individual to thereby select appropriatetherapeutic or prophylactic agents for that individual (referred toherein as “pharmacogenomics”). Pharmacogenomics allows for the selectionof agents (e.g., drugs) for therapeutic or prophylactic treatment of anindividual based on the genotype of the individual (e.g., the genotypeof the individual examined to determine the ability of the individual torespond to a particular agent.) Yet another aspect of the inventionpertains to monitoring the influence of agents (e.g., drugs, compounds)on hypoxic conditions, cancer or infection in clinical trials.

Thus, the diagnostic methods described herein can furthermore beutilized to identify subjects having or at risk of developing a diseaseor disorder associated with hypoxic conditions, cancer or infection.Furthermore, the prognostic assays described herein can be used todetermine whether a subject should be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with hypoxic conditions, cancer or infection.

The selection of a light-generating polypeptide moiety of thelight-generating fusion protein should be done so as to produce lightcapable of penetrating animal tissue such that it can be detectedexternally in a non-invasive manner. The ability of light to passthrough a medium such as animal tissue (composed mostly of water) isdetermined primarily by the light's intensity and wavelength.

The more intense the light produced in a unit volume, the easier thelight will be to detect. The intensity of light produced in a unitvolume depends on the spectral characteristics of individuallight-generating polypeptide moieties, and on the concentration of thosemoieties in the unit volume. Accordingly, schemes that place a highconcentration of light-generating polypeptide moieties in or on anentity (such as high-efficiency loading of a liposome or high-levelexpression of a light-generating fusion protein in a cell) typicallyproduce brighter light-generating fusion proteins (LGPs), which areeasier to detect through deeper layers of tissue, than schemes whichconjugate, for example, only a single LGM onto each entity.

A second factor governing detectability through a layer of tissue is thewavelength of the emitted light. Water may be used to approximate theabsorption characteristics of animal tissue, since most tissues arecomposed primarily of water. It is well known that water transmitslonger-wavelength light (in the red range) more readily than it doesshorter wavelength light.

Accordingly, light-generating polypeptide moieties which emit light inthe range of yellow to red (550-1100 nm) are typically preferable tothose which emit at shorter wavelengths. However, excellent results canbe achieved in practicing the present invention with LGMs that emit inthe range of 486 nm, despite the fact that this is not an optimalemission wavelength.

Fluorescence-Based Moieties.

Because fluorescent molecules require input of light in order toluminesce, their use in the invention may be more involved than the useof bioluminescent molecules. Precautions are typically taken to shieldthe excitatory light so as not to contaminate the fluorescence photonsignal being detected from the subject. Obvious precautions include theplacement of an excitation filter at the radiation source. Anappropriately-selected excitation filter blocks the majority of photonshaving a wavelength similar to that of the photons emitted by thefluorescent moiety. Similarly, a barrier filter is employed at thedetector to screen out most of the photons having wavelengths other thanthat of the fluorescence photons. Filters such as those described abovecan be obtained from a variety of commercial sources, including OmegaOptical, Inc. (Brattleboro, Vt.).

Alternatively, a laser producing high intensity light near theappropriate excitation wavelength, but not near the fluorescenceemission wavelength, can be used to excite the fluorescent moieties. Anx-y translation mechanism may be employed so that the laser can scan thesubject, for example, as in a confocal microscope.

As an additional precaution, the radiation source may be placed behindthe subject and shielded, such that the only radiation photons reachingthe site of the detector are those that pass all the way through thesubject. Furthermore, detectors may be selected that have a reducedsensitivity to wavelengths of light used to excite the fluorescentmoiety.

An advantage of small fluorescent molecules is that they are less likelyto interfere with the bioactivity of the entity to which they areattached than would a larger light-generating moiety. In addition,commercially-available fluorescent molecules can be obtained with avariety of excitation and emission spectra that are suitable for usewith the present invention. For example, Molecular Probes (Eugene,Oreg.) sells a number of fluorophores, including Lucifer Yellow (abs. at428 nm, and emits at 535 nm) and Nile Red (abs. at 551 nm and emits at636 nm). Further, the molecules can be obtained derivatized with avariety of groups for use with various conjugation schemes (e.g., fromMolecular Probes).

Bioluminescence-Based Moieties.

The subjects of chemiluminescence (luminescence as a result of achemical reaction) and bioluminescence (visible luminescence from livingorganisms) have, in many aspects, been thoroughly studied.

An advantage of bioluminescent moieties over fluorescent moieties isthat there is virtually no background in the signal. The only lightdetected is light that is produced by the exogenous bioluminescentmoiety. In contrast, the light used to excite a fluorescent moleculeoften results in the fluorescence of substances other than the intendedtarget. This is particularly true when the “background” is as complex asthe internal environment of a living animal.

Ligands include, in a preferred embodiment, enzymes which are capable ofmodifying a light-generating polypeptide moiety such that it does orceases to emit light when modified. In use, this happens when, e.g., thelight-generating fusion protein comes in contact with a ligand on anentity or produced by the entity, and the ligand binds to the ligandbinding site, altering the light-generating properties of thelight-generating polypeptide moiety. Examples include the following.

In an especially useful embodiment of the present invention, alight-generating fusion protein comprising a binding site for an E3ubiquitin ligase and a light-generating polypeptide moiety capable ofbeing modified by E3 at a modification site can be used for diagnosticand treatment purposes. Ubiquitin ligases (e.g., E3) bind to colinear‘T’ (see FIG. 1) present on their substrates. Examples include the SCF(Skp1/Cdc53/F-box) ubiquitin ligases, the VBC (pVHL/elongin B/elongin C)ubiquitin ligase, and the MDM2 ubiquitin ligase. It is alreadyestablished that light-generating proteins such as GFP and luciferasecan be fused to heterologous polypeptides without loss of activity. Insome embodiments, the light-generating polypeptide moiety may bemodified to include a surface exposed lysine residue accessible as aubiquitin acceptor site. In a First embodiment, a light-generatingfusion protein of the invention may be used to monitor ischemia inliving tissues and animals. The First embodiment comprises a polypeptidederived from the HIFα (hypoxia-inducible factor 1α) which acts as abinding site for VBC. This binding site is only recognized by VBC in thepresence of oxygen as a result of proline hydroxylation. Thelight-generating fusion protein of the First embodiment isadvantageously stable in hypoxic cells, but unstable in well oxygenatedcells. In a related embodiment, the HIF-derived polypeptide describedabove may be fused to a suicide moiety such as a protein (such as HSV TKor an adenoviral protein like E1A), which can be used in selectivekilling of ischemic cells (such as in a solid tumor).

Binding and/or modification sites are well-known in the art for avariety of kinases including cyclin-dependent kinases, ATM/ATR, JNKkinases, and receptor tyrosine kinases. In one embodiment, alight-generating fusion protein may be fused to a binding site for akinase of interest. In some embodiments, it is useful to introduce oneor more binding sites for a selected kinase into the light-generatingfusion protein. By way of example, since phosphorylated serine,threonine, and tyrosine, by virtue of their negative charges, frequentlymimic aspartic acid and/or glutamic acid residues, individual asparticacid and glutamic acid residues are replaced with serine, threonine, ortyrosine (in the context of the kinase modification site). Suchsubstitutions may be made singly and in combination. In anotherembodiment, the modification site can be empirically determined bycarrying out a linker scan of the light-generating protein using alinker encoding the kinase modification site. In yet another embodimentthe light-generating polypeptide moiety is mutagenized (either random ortargeted mutagenesis) to generate modification sites in which thelight-generating polypeptide moiety is selectively inactivated oractivated by the kinase; this mutagenesis is performed using cells (suchas yeast) rendered conditional for the kinase. In still yet anotherembodiment, a kinase modification site is designed in silico based oncomparison of the binding site of the selected kinase to the primarysequence of the light-generating polypeptide moiety, coupled withknowledge of the three dimensional structure of the light-generatingpolypeptide moiety.

In an additional embodiment described below, a light-generatingpolypeptide moiety is fused to a polypeptide recognized by a cyclin/cdk2complex. This enzyme phosphorylates serine or threonine with an absoluterequirement for proline in the +1 position. By way of example, GFPcontains 13 proline residues including one threonine-proline site andtwo aspartic acid-proline sites. In one embodiment, a light-generatingprotein (e.g., GFP) is fused to a cyclin/cdk2 binding site such asY-Lys-X₁-Leu-K-X₂-Leu-Y′ (SEQ ID NO.9). The light-generating protein isphosphorylated and inactivated by cyclin/cdk2, providing a detectablesignal which is selectively off in the presence of cyclin/cdk2. Inanother embodiment, the light-generating polypeptide moiety is mutatedso that it is not phosphorylated and activated by cyclin/cdk2. Anexample of this mutation would be to mutate the two asparticacid-proline sites to serine (or threonine)-prolines.

In this additional embodiment, the ligand binding site is a polypeptiderecognized by a cyclin/cdk2 complex, e.g., comprising the amino acidsequence Y-Lys-X₁-Leu-K-X₂-Leu-Y′, wherein X₁, X₂, are independently anyone or more amino acids; and Y and Y′ are independently present orabsent and, if present, independently comprise a peptide having from 1to 600 amino acids.

An example of such a target peptide is RB: PKPLKKLRFD (SEQ ID NO: 1). Ina related aspect of this additional embodiment, the ligand binding sitecomprises the amino acid sequence Y-X₁-Arg-Arg-Leu-Y′, wherein X₁ is Lysor Cys; and Y and Y′ are independently present or absent and, ifpresent, independently comprise a peptide having from 1 to 600 aminoacids. Two non-limiting examples of the target peptide of this relatedaspect of this additional embodiment are: E2F1: GRPPVKRRLDLE (SEQ ID NO:2); derived from the E2F1 protein, and p21: CCSKACRRLFGP (SEQ ID NO: 3),derived from the p21 protein.

The fusion protein of this additional embodiment can be produced bystandard recombinant DNA techniques, discussed supra.

The ligand binding site of the light-generating fusion protein of thisadditional embodiment is derived from the retinoblastoma (RB) protein.Adams, et al., 1999 Mol. Cell Biol. 19:1068 describes RB protein. An RBpolypeptide comprises 928 amino acid residues. As described in moredetail below, this ligand binding site comprises a unique cyclin bindingdomain derived from RB. In the light-generating fusion protein describedabove, where present, Y comprises between 1 and 900 amino acid residues,preferably corresponding to the sequence of “N-terminal” RB amino acidresidues 1-868. Thus, in a preferred embodiment Y can represent anysequence of N-terminal amino acids of RB, for example residues 794-829,and so on up to and including residues 1-868. Y′ can also comprisebetween 1 and 600 amino acid residues, preferably corresponding to thesequence of “C-terminal” RB amino acid residues 879-928. Thus, in apreferred embodiment, Y′ can represent any sequence of C-terminal aminoacids of RB, for example 879-910, and so on up to and including residues879-928. Y and Y′ can also contain conservative substitutions of aminoacids present in the N-terminal and C-terminal RB sequences describedabove. In a further preferred embodiment, Y and Y′ are absent.

The light-generating fusion protein of this additional embodimentprovides a way for detecting “cancerous tissue” or tissue subject toaberrant cell proliferation and therefore at risk for cancer. Inaddition to tissue that becomes cancerous due to an in situ neoplasm,for example, the light-generating fusion protein also provides a methodof detecting cancerous metastatic tissue present in distal organs and/ortissues. Thus such tissue may be detected by contacting tissue suspectedof being cancerous with the light-generating fusion protein underappropriate conditions to cause the phosphorylation of thelight-generating fusion protein in cancerous tissue, thereby detectingthe presence of cancerous tissue. The ligand binding site of thelight-generating fusion protein of this embodiment provides a bindingsite for cyclin-cyclin-dependent kinase (cdk) complexes whichphosphorylate the protein, causing it to emit or not emit light in thepresence of active cycle-cdk complexes. Under appropriate conditions,the light-generating fusion protein will therefore be phosphorylated orinactive in tissue that is not cancerous, and unphosphorylated andpreserving its light-emitting properties in tissue that is cancerous,e.g. primary and metastatic tissue.

In another useful embodiment, a light-generating polypeptide moiety isfused to a polypeptide recognized by a cyclin/cdk2 complex. This enzymephosphorylates serine or threonine with an absolute requirement forproline in the +1 position. By way of example, GFP contains 13 prolineresidues including one threonine-proline site and two asparticacid-proline sites. In one embodiment, a light-generating protein (e.g.,GFP) is fused to a cyclin/cdk2 binding site such asY-Lys-X₁-Leu-K-X₂-Leu-Y′ (SEQ ID NO.9). The light-generating protein isphosphorylated and inactivated by cyclin/cdk2, providing a detectablesignal which is selectively off in the presence of cyclin/cdk2. Inanother embodiment, the light-generating polypeptide moiety is mutatedso that it is not phosphorylated and activated by cyclin/cdk2. Anexample of this mutation would be to mutate the two asparticacid-proline sites to serine (or threonine)-prolines.

Phosphorylation is one of the most important ways to posttranslationallymodify proteins, and it regulates diverse cell physiological processes(transport, proliferation, differentiation). For example,phosphorylation is involved in all phases of cell division: intransition from G1 to S phase, progression of cells during S phase andentry into M phase. The physiological function of oncoproteins and tumorsuppressor proteins that are involved in gene expression and replicationare also regulated by phosphorylation. Many growth factors and theirreceptors are encoded by oncogenes which are mutated or overexpressed ina variety of human tumors. Mutation or overexpression of these oncogenesleads to unchecked cell division, and transformation of normal cells tomalignant. In an embodiment of the present invention, a light-generatingfusion protein may include a phosphatase binding site and a modificationsite, such as a phosphorylated amino acid residue capable of beingmodified by a protein “phosphatase”.

For most proteases, the enzyme binding site and modification site arelargely congruent and can be encompassed in short peptides. In anembodiment, a light-generating fusion protein may include a proteasebinding site and a modification site capable of being cleaved by theprotease. The binding site and modification site may be the same site orin two discrete regions. In one embodiment, wherein the binding andmodification site are congruent, linker scanning mutagenesis of a givenlight-generating protein is carried out using a linker that encodes thecongruent binding/modification site. Resulting mutants are thoselight-generating fusion proteins containing a binding/modification sitethat preserves light-emitting activity in protease deficient cells andexhibits protease sensitivity in vitro and in vivo. In the Thirdembodiment described herein, a light-generating fusion proteincontaining an HIV protease site is especially useful for monitoring thepresence or absence of HIV (in this case, with HIV-positive cells notemitting light). In an alternative embodiment, the light-generatingfusion protein is fused via a linker to a protein that inhibits thelight-emitting activity of the light-generating polypeptide moiety. Thelinker includes a binding/modification site for an HIV protease. Thus,cleavage at the binding/modification site should remove the inhibitingprotein moiety, thus yielding a positive signal in cells that are HIVpositive.

As shown generally in FIG. 1A, an embodiment of the fusion protein ofthe invention contains a ligand binding site “T”, a reporter domain(e.g., light-generating polypeptide moiety) “R”, and a modification site“X”. Binding of enzyme “E” (the ligand) to target site “T” results in amodification of modification site “X”, which causes the reporter domain“R” to either emit or not emit light.

The site “T” and modification site “X” may be separate and in cis on thefusion protein, as shown in FIG. 1B. Either “T” or “X” can be proximalor distal to the amino terminus of the fusion protein.

In another embodiment shown in FIG. 1C, wherein the “T” and the “X” ofthe fusion protein are congruent within a domain of the fusion protein.In related embodiments, the congruent “T/X” can be at the aminoterminus, the carboxy terminus, or at neither terminus of the fusionprotein.

In a further embodiment shown in FIG. 1D, the “T” is on a proteinassociated with the fusion protein containing the “X”. In a relatedembodiment, target site “T” is on the fusion protein and modificationsite “X” is on a protein that is physically associated with the fusionprotein, wherein modification of the associated protein results in thefusion protein either emitting or not emitting light. The depiction ofsite “T” and modification site “X” are not intended to be limiting. “T”can be at the amino terminus, the carboxy terminus, or at neitherterminus of the fusion protein, and “X” can be at the amino terminus,the carboxy terminus, or at neither terminus of the associated protein.

The invention also includes a fusion protein in which the ligand bindingsite sequence “T” is unknown. In this embodiment, e.g., as shown in FIG.2A, the fusion protein includes a first hetero- or homo-dimerizationdomain (“HD1”). An enzyme “E” capable of modifying modification site “X”on the fusion protein is fused to a second hetero- or homo-dimerizationdomain “HD2” that interacts with “HD1”. In a related embodiment, thetargeting site “T” for an enzyme “E” can be generated by inserting oneor more polypeptide sequences derived from a random or non-randompeptide library into the fusion protein.

In a related embodiment (FIG. 2B), a binding domain of a protein “A”containing an enzyme target site “T” interacts with a binding domain “B”of a fusion protein, which results in enzyme “E” modifying the reporterdomain “R” of fusion protein “B” at modification site “X” such that thefusion protein does or does not emit light or cause light to be emitted.In the First embodiment, the ligand binding site comprises a polypeptidederived from the HIF1α (hypoxia-inducible factor 1α) which acts as abinding site for VBC, e.g., the amino acid sequenceY-X₁-Leu-X₂-Pro_(h)-X₃-X₄-X₅-X₆-Y′, wherein Pro_(h) is hydroxylatedproline; X₁, X₂, X₃, X₄, X₅, and X₆ are amino acids selected so as tonot modify or alter VHL binding properties. X₁, X₂, X₄, X₅, and X₆ aredesirably independently Gly, Ala, Val, Leu, Ile, Pro, Met, Phe, or Trp,and X₃ is desirably Ser, Thr, or Tyr; and Y and Y′ are independentlypresent or absent and, if present, independently comprise a peptidehaving from 1 to 600 amino acids.

In a preferred embodiment, the ligand binding site comprises the aminoacid sequence corresponding to the N-terminal residues 1-600 of HIF1α,wherein either or both of residues 402 and 564 are proline orhydroxylated proline. In a more preferred embodiment, the ligand bindingsite comprises an 80 to 120, 20 to 30, 12 to 14, or 4 to 12 amino acidsequence corresponding to the residues adjacent to and/or surroundingresidue 402 and/or 564, inclusive, of HIF1α, wherein residues 402 and/or564 is proline or hydroxylated proline. “Residues adjacent to and/orsurrounding” is meant to include the relevant sequence of HIF1α eitherbefore, after, or flanking, the specified residue, e.g., residue 564.

Such proteins may be used effectively as oxygen-sensing proteins. Thefusion proteins may be produced by standard recombinant DNA techniques.For example, DNA fragments coding for the different polypeptidesequences are ligated together in-frame in accordance with conventionaltechniques, e.g., by employing blunt-ended or stagger-ended termini forligation, restriction enzyme digestion to provide for appropriatetermini, filling-in of cohesive ends as appropriate, alkalinephosphatase treatment to avoid undesirable joining, and enzymaticligation. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers that give rise to complementary overhangs betweentwo consecutive gene fragments that can subsequently be annealed andreamplified to generate a chimeric gene sequence (see, e.g., Ausubel etal. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons,1992).

The ligand binding site noted above is derived from HIF1α protein. U.S.Pat. No. 6,222,018, describes HIF protein and its preparation insubstantially pure form. HIF is composed of subunits HIF1α and anisoform HIF1β. HIF1α polypeptide comprises 826 amino acid residues. Asdescribed in more detail below, the ligand binding sites of thisparticular fusion protein comprises a unique ubiquitin ligase bindingdomain derived from HIF1α. In this fusion protein, where present, Ycomprises between 1 and 600 amino acid residue, preferably correspondingto the sequence of “N-terminal” HIF1α amino acid residues 1-555. Thus,in a preferred embodiment Y can represent any sequence of N-terminalamino acids of HIF1α, for example residues 554-555, 553-555, 552-555,and so on up to and including residues 1-555. Y′ can also comprisesbetween 1 and 600 amino acid residues, preferably corresponding to thesequence of “C-terminal” HIF1α amino acid residues 576-826. Thus, in apreferred embodiment, Y′ can represent any sequence of C-terminal aminoacids of HIF1α, for example 576-577, 576-578, 576-579, and so on up toand including residues 576-826. Y and Y′ can also contain conservativesubstitutions of amino acids present in the N-terminal and C-terminalHIF1α sequences described above. In a preferred embodiment of the ligandbinding site defined above, Y and Y′ are absent. In a further preferredembodiment, X₁ is Met, X₂ is Leu, X₃ is Ala, X₄ is Tyr, X₅ is Pro, andX₆ is Met. In a particularly preferred embodiment of the fusion protein,the ligand binding site has the amino acid sequenceAsp-Leu-Asp-Leu-Glu-Met-Leu-Ala-Pro_(h)-Tyr-Ile-Pro-Met-Asp-Asp-Asp-Phe-Gln-Leu-Arg,corresponding to HIF1α amino acid residues 556-575, with a hydroxylatedproline at amino acid residue 564.

The invention also provides a nucleic acid molecule encoding the fusionprotein or polypeptide of the invention. (As used herein, the termspolypeptide and protein are interchangeable). An “isolated” nucleic acidmolecule is one that is separated from other nucleic acid molecules thatare present in the natural source of the nucleic acid. Examples ofisolated nucleic acid molecules include, but are not limited to,recombinant DNA molecules contained in a vector, recombinant DNAmolecules maintained in a heterologous host cell, partially orsubstantially purified nucleic acid molecules, and synthetic DNA or RNAmolecules. Moreover, an “isolated” nucleic acid molecule, such as a cDNAmolecule, can be substantially free of other cellular material orculture medium when produced by recombinant techniques, or of chemicalprecursors or other chemicals when chemically synthesized.

A nucleic acid of the invention can be amplified using cDNA, mRNA oralternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to the nucleotide sequencescan be prepared by standard synthetic techniques, e.g., using anautomated DNA synthesizer. The present invention therefore also providesa vector comprising the nucleic acid of the invention. In a preferredembodiment, the vector further comprises a promoter operably linked tothe nucleic acid molecule. In a further preferred embodiment theinvention provides a cell containing the vector of the invention.

The light-generating fusion protein of, e.g., the First embodimentprovides a means for detecting hypoxic tissue or tissue subject tochronic hypoxia and therefore at risk for ischemia. In addition totissue that becomes ischemic due to occurrence of a stroke, heart attackor embolism, for example, the fusion protein also provides a method ofdetecting ischemic tissue present in tumors. Thus the invention may beused to detect such tissue by contacting tissue suspected of beinghypoxic with the light-generating fusion protein under appropriateconditions to cause the ubiquitination of the fusion protein in normoxictissue, thereby detecting the presence of hypoxic tissue. As describedmore fully below, the ligand binding site of the light-generating fusionprotein also provides a binding site for ubiquitin ligases which destroythe protein in the presence of oxygen. Under appropriate conditions, thelight-generating fusion protein of the invention will therefore be“unstable” or destroyed in tissue that is not hypoxic, i.e., that iswell oxygenated, and “stable” or preserving its light-emittingproperties in tissue that is hypoxic, i.e. that lacks sufficient oxygen.

The Third embodiment provides a means for detecting “infected tissue” ortissue subject to contact with infectious agents and therefore at riskfor infection, e.g., monitoring the presence or absence of HIV. Theligand binding site of the Third embodiment comprises a binding site fora protease, such as an infection-associated protease. For example,protease cleavage sites of the human immunodeficiency virus (HIV-1)protein precursor Pr55 (gag) protein include p2/NC, NC/p1, and NC/TFP.

The ligand binding site of the light-generating fusion protein of thisThird embodiment may be derived from the HIV-1 protein. One non-limitingexample of the target peptide of this Third embodiment is:Y-GSGIF*LETSL-Y′ (See Beck et al., (2000) Virology 274(2):391-401). Yand Y′ are independently present or absent and, if present,independently comprise a peptide having from 1 to 600 amino acids, and“*” indicates the cleavage site of the fusion protein by a protease.

In addition to tissue that becomes infected due to an acute or chronicinfection by an infectious agent, for example, the light-generatingfusion protein may be used in detecting infected tissue present indistal organs and/or tissues, e.g., by contacting tissue suspected ofbeing infected with the light-generating fusion protein of the inventionunder appropriate conditions to cause the proteolysis of thelight-generating fusion protein in infected tissue, thereby detectingthe presence of infected tissue. The ligand binding site of thelight-generating fusion protein of this Third embodiment provides abinding site for a protease which degrades or modifies the protein,causing it to emit or not emit light in the presence of one or moreproteases. Under appropriate conditions, the light-generating fusionprotein will therefore be proteolyzed or inactive in tissue that isinfected (although there may be cases where proteolysis leads to lightgeneration), and unproteolyzed and preserving its light-emittingproperties in tissue that is infected.

A Fourth embodiment of a light-generating fusion protein of theinvention comprises a light-generating protein moiety and a ligandbinding site, wherein the ligand binding site comprises an amino acidsequence capable of binding to an “associated” protein. The associationcan occur by covalent or non-covalent binding. This associated proteinmay itself be a light-generating fusion protein, comprising a bindingpolypeptide capable of binding to the light-emitting light-generatingfusion protein and an enzyme capable of modifying the light-emittinglight-generating fusion protein.

A non-limiting example of the target peptide of this Fourth embodimentis: HD1: WFHGKLSR (Amino acids 488-495 of Accession No. P29353, humanSHC1; SEQ ID NO: 4). This target polypeptide contains an SH2 domain.Therefore, any associated protein with an SH2 domain should interactwith the target peptide of the Fourth embodiment. A non-limiting exampleof the binding peptide of the associated protein of this Fourthembodiment is: HD2: WNVGSSNR (Amino acids 624-631 of Accession No.P27986; human PI3K p85 subunit; SEQ ID NO: 5).

Another non-limiting example of the Fourth embodiment would be to fusethe FK506 binding protein (FKBP12) domain moiety to GFP and the FRAPdomain moiety to Skp1 or elonginC. Therefore in the presence ofrapamycin, which promotes the high affinity interaction of FKBP12 andFRAP, the core E3 ligase machinery would bind to and destroy GFP,eliminating the bioluminescence wherever rapamycin is present. Theligand binding site of the light-generating fusion protein of thisFourth embodiment is derived from the human SHC1 proteinprotein. SeePelicci et al., (1992) Cell 70:93-104, describes the SHC1 protein withan SH2 domain which is implicated in mitogenic signal transduction. Thelight-generating fusion protein of this Fourth embodiment provides ameans for detecting enzymatic activity where the enzyme binding site isundefined.

The pharmaceutical compositions of the invention comprise the novelagents combined with a pharmaceutically acceptable carrier. The term“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. Suitable carriers aredescribed in the most recent edition of Remington's PharmaceuticalSciences, a standard reference text in the field, which is incorporatedherein by reference. Preferred examples of such carriers or diluentsinclude, but are not limited to, water, saline, finger's solutions,dextrose solution, and 5% human serum albumin. Liposomes and non-aqueousvehicles such as fixed oils may also be used. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfate; chelating agents such as ethylenediaminetetraacetic acid(EDTA); buffers such as acetates, citrates or phosphates, and agents forthe adjustment of tonicity such as sodium chloride or dextrose. The pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™(BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixtures thereofThe proper fluidity can be maintained, for example, by the use of acoating such as lecithin, by the maintenance of the required particlesize in the case of dispersion and by the use of surfactants.

Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The invention includes entities which may have been modified orconjugated to include a light-generating fusion protein of theinvention. Such conjugated or modified entities are referred to aslight-emitting entities, or simply conjugates. The conjugates themselvesmay take the form of, for example, molecules, macromolecules, particles,microorganisms, or cells. The methods used to conjugate alight-generating fusion protein to an entity depend on the nature of thelight-generating fusion protein and the entity. Exemplary conjugationmethods are discussed in the context of the entities described below.

Small Molecules.

Small molecule entities which may be useful in the present inventioninclude compounds which specifically interact with a pathogen or anendogenous ligand or receptor. Examples of such molecules include, butare not limited to, drugs or therapeutic compounds; toxins, such asthose present in the venoms of poisonous organisms, including certainspecies of spiders, snakes, scorpions, dinoflagellates, marine snailsand bacteria; growth factors, such as NGF, PDGF, TGF and TNF; cytokines;and bioactive peptides.

The small molecules are preferably conjugated to light-generating fusionproteins in a way that that the bioactivity of the small molecule is notsubstantially affected. Conjugations are typically chemical in nature,and can be performed by any of a variety of methods known to thoseskilled in the art.

Small molecules conjugated to light-generating fusion proteins of thepresent invention may be used either in animal models of humanconditions or diseases, or directly in human subjects to be treated. Forexample, a small molecule which binds with high affinity to receptorexpressed on tumor cells may be used in an animal model to localize andobtain size estimates of tumors, and to monitor changes in tumor growthor metastasis following treatment with a putative therapeutic agent.Such molecules may also be used to monitor tumor characteristics, asdescribed above, in cancer patients.

Macromolecules.

Macromolecules, such as polymers and biopolymers, constitute anotherexample of entities useful in practicing the present invention.Exemplary macromolecules include antibodies, antibody fragments,light-generating fusion proteins and certain vector constructs.

Antibodies or antibody fragments, purchased from commercial sources ormade by methods known in the art, can be used to localize their antigenin a mammalian subject by conjugating the antibodies to alight-generating polypeptide moiety, administering the conjugate to asubject by, for example, injection, allowing the conjugate to localizeto the site of the antigen, and imaging the conjugate.

Antibodies and antibody fragments have several advantages for use asentities in the present invention. By their nature, they constitutetheir own targeting moieties. Further, their size makes them amenable toconjugation with several types of light-generating fusion proteins,including small fluorescent molecules and fluorescent and bioluminescentproteins, yet allows them to diffuse rapidly relative to, for example,cells or liposomes.

The light-generating fusion proteins can be conjugated directly to theantibodies or fragments, or indirectly by using, for example, afluorescent secondary antibody. Direct conjugation can be accomplishedby standard chemical coupling of, for example, a fluorophore to theantibody or antibody fragment, or through genetic engineering. Chimeras,or fusion proteins, can be constructed which contain an antibody orantibody fragment coupled to a fluorescent or bioluminescent protein.For example, Casadei, et al., describe a method of making a vectorconstruct capable of expressing a fusion protein of aequorin and anantibody gene in mammalian cells.

Conjugates containing antibodies can be used in a number of applicationsof the present invention. For example, a labeled antibody directedagainst E-selection, which is expressed at sites of inflammation, can beused to localize the inflammation and to monitor the effects of putativeanti-inflammatory agents.

Vector constructs by themselves can also constitute macromolecularentities applicable to the present invention. For example, a eukaryoticexpression vector can be constructed which contains a therapeutic geneand a gene encoding a light-generating molecule under the control of aselected promoter (i.e., a promoter which is expressed in the cellstargeted by the therapeutic gene). Expression of the light-generatingmolecule, assayed using methods of the present invention, can be used todetermine the location and level of expression of the therapeutic gene.This approach may be particularly useful in cases where the expressionof the therapeutic gene has no immediate phenotype in the treatedindividual or animal model.

Viruses.

Another entity useful for certain aspects of the invention are viruses.As many viruses are pathogens which infect mammalian hosts, the virusesmay be conjugated to a light-generating fusion protein and used to studythe initial site and spread of infection. In addition, viruses labeledwith a light-generating fusion protein may be used to screen for drugswhich inhibit the infection or the spread of infection.

A virus may be labeled indirectly, either with an antibody conjugated toa light-generating fusion protein, or by, example, biotinylating virionsas known in the art and then exposing them to streptavidin linked to adetectable moiety, such as a fluorescent molecule.

Alternatively, virions may be labeled directly with a fluorophore likerhodamine, using methods known in the art. The virus can also begenetically engineered to express a light-generating fusion protein.Labeled virus can be used in animal models to localize and monitor theprogression of infection, as well as to screen for drugs effective toinhibit the spread of infection. For example, while herpes virusinfections are manifested as skin lesions, this virus can also causeherpes encephalitis. Such an infection can be localized and monitoredusing a virus labeled by any of the methods described above, and variousantiviral agents can be tested for efficacy in central nervous system(CNS) infections.

Particles.

Particles, including beads, liposomes and the like, constitute anotherentity useful in the practice of the present invention. Due to theirlarger size, particles may be conjugated with a larger number oflight-generating fusion proteins than, for example, can small molecules.This results in a higher concentration of light emission, which can bedetected using shorter exposures or through thicker layers of tissue. Inaddition, liposomes can be constructed to contain an essentially puretargeting moiety, or ligand, such as an antigen or an antibody, on theirsurface. Further, the liposomes may be loaded with, for example,light-generating protein molecules, to relatively high concentrations.

Furthermore, two types of liposomes may be targeted to the same celltype such that light is generated only when both are present. Forexample, one liposome may carry luciferase, while the other carriesluciferin. The liposomes may carry targeting moieties, and the targetingmoieties on the two liposomes may be the same or different. Viralproteins on infected cells can be used to identify infected tissues ororgans. Cells of the immune system can be localized using a single ormultiple cell surface markers.

The liposomes are preferably surface-coated, e.g., by incorporation ofphospholipid-polyethyleneglycol conjugates, to extend blood circulationtime and allow for greater targeting via the bloodstream. Liposomes ofthis type are well known.

Cells.

Cells, both prokaryotic and eukaryotic, constitute another entity usefulin the practice of the present invention. Like particles, cells can beloaded with relatively high concentrations of light-generating moieties,but have the advantage that the light-generating moieties can beprovided by, for example, a heterologous genetic construct used totransfect the cells. In addition, cells can be selected that express“targeting moieties”, or molecules effective to target them to desiredlocations within the subject. Alternatively, the cells can betransfected with a vector construct expressing an appropriate targetingmoiety.

The cell type used depends on the application. For example, bacterialcells can be used to study the infective process, and to evaluate theeffects of drugs or therapeutic agents on infective process with a highlevel of temporal and spatial resolution. Bacterial cells constituteeffective entities. For example, they can be easily transfected toexpress a high levels of a light-generating fusion protein, as well ashigh levels of a targeting protein. In addition, it is possible toobtain E. coli libraries containing bacteria expressing surface-boundantibodies which can be screened to identify a colony expressing anantibody against a selected antigen (Stratagene, La Jolla, Calif.).Bacteria from this colony can then be transformed with a second plasmidcontaining a gene for a light-generating protein, and transformants canbe utilized in the methods of the present invention, as described above,to localize the antigen in a mammalian host.

Pathogenic bacteria can be conjugated to a light-generating fusionprotein and used in an animal model to follow the infection process invivo and to evaluate potential anti-infective drugs, such as newantibiotics, for their efficacy in inhibiting the infection.

Eukaryotic cells are also useful as entities in aspects of the presentinvention. Appropriate expression vectors, containing desired regulatoryelements, are commercially available. The vectors can be used togenerate constructs capable of expressing desired light-generatingproteins in a variety of eukaryotic cells, including primary culturecells, somatic cells, lymphatic cells, etc. The cells can be used intransient expression studies, or, in the case of cell lines, can beselected for stable transformants.

Expression of the light-generating protein in transformed cells can beregulated using any of a variety of selected promoters. For example, ifthe cells are to be used as light-emitting entities targeted to a sitein the subject by an expressed ligand or receptor, aconstitutively-active promoter, such as the CMV or SV40 promoter may beused. Cells transformed with such a construct can also be used to assayfor compounds that inhibit light generation, for example, by killing thecells.

Alternatively, the transformed cells may be administered such theybecome uniformly distributed in the subject, and express thelight-generating fusion protein only under certain conditions, such asupon infection by a virus or stimulation by a cytokine. Promoters thatrespond to factors associated with these and other stimuli are known inthe art. In a related aspect, inducible promoters, such as the Tetsystem can be used to transiently activate expression of thelight-generating protein.

For example, CD4+ lymphatic cells can be transformed with a constructcontaining tat-responsive HIV LTR elements, and used as an assay forinfection by HIV. Cells transformed with such a construct can beintroduced into SCID-hu mice and used as model for human HIV infectionand AIDS.

Tumor cell lines transformed to express the light-generating fusionprotein, for example, with a constitutively-active promoter, may be usedto monitor the growth and metastasis of tumors. Transformed tumor cellsmay be injected into an animal model, allowed to form a tumor mass, andthe size and metastasis of the tumor mass monitored during treatmentwith putative growth or metastasis inhibitors. Tumor cells may also begenerated from cells transformed with constructs containing regulatablepromoters, whose activity is sensitive to various infective agents, orto therapeutic compounds.

Cell Transformation.

Transformation methods for both prokaryotic cells and eukaryotic cellsare well known in the art. Vectors containing the appropriate regulatoryelements and multiple cloning sites are widely commercially available(e.g., Stratagene, La Jolla, Calif., or Clontech, Palo Alto, Calif.).

In another aspect, the present invention includes transgenic animalscontaining a heterologous gene construct encoding a light-generatingfusion protein or complex of proteins. The construct is driven by aselected promoter, and can include, for example, various accessoryproteins required for the functional expression of the light-generatingprotein, as well as selection markers and enhancer elements.

Activation of the promoter results in increased expression of the genesencoding the light-generating fusion proteins and accessory proteins.Activation of the promoter is achieved by the interaction of a selectedbiocompatible entity, or parts of the entity, with the promoterelements. If the activation occurs only in a part of the animal, onlycells in that part will express the light-generating protein.

Light-generating fusion proteins are typically administered to a subjectby any of a variety of methods, allowed to localize within the subject,and imaged. Since the imaging, or measuring photon emission from thesubject, may last up to tens of minutes, the subject is desirablyimmobilized during the imaging process. Imaging of the light-generatingpolypeptide moiety involves the use of, e.g., a photodetector capable ofdetecting extremely low levels of light—typically single photonevents—and integrating photon emission until an image can beconstructed. Examples of such sensitive photodetectors include devicesthat intensify the single photon events before the events are detectedby a camera, and cameras (cooled, for example, with liquid nitrogen)that are capable of detecting single photons over the background noiseinherent in a detection system.

Once a photon emission image is generated, it is typically superimposedon a “normal” reflected light image of the subject to provide a frame ofreference for the source of the emitted photons (i.e., localize thelight-generating fusion proteins with respect to the subject). Such a“composite” image is then analyzed to determine the location and/oramount of a target in the subject.

Light-generating fusion proteins that have localized to their intendedsites in a subject may be imaged in a number of ways. Guidelines forsuch imaging, as well as specific examples, are described below.

Localization of Light-Generating Fusion Proteins.

In the case of “targeted” conjugates, that is, conjugates which containa targeting moiety—a molecule or feature designed to localize theconjugate within a subject or animal at a particular site or sites,localization refers to a state when an equilibrium between bound,“localized”, and unbound, “free” entities within a subject has beenessentially achieved. The rate at which such an equilibrium is achieveddepends upon the route of administration. For example, a conjugateadministered by intravenous injection to localize thrombi may achievelocalization, or accumulation at the thrombi, within minutes ofinjection. On the other hand, a conjugate administered orally tolocalize an infection in the intestine may take hours to achievelocalization.

Alternatively, localization may simply refer to the location of theentity within the subject or animal at selected time periods after theentity is administered. In a related aspect, localization of, forexample, injected tumors cells expressing a light-generating moiety, mayconsist of the cells colonizing a site within the animal and forming atumor mass.

By way of another example, localization is achieved when an entitybecomes distributed following administration. For example, in the caseof a conjugate administered to measure the oxygen concentration invarious organs throughout the subject or animal, the conjugate becomes“localized”, or informative, when it has achieved an essentiallysteady-state of distribution in the subject or animal.

In all of the above cases, a reasonable estimate of the time to achievelocalization may be made by one skilled in the art. Furthermore, thestate of localization as a function of time may be followed by imagingthe light-emitting conjugate according to the methods of the invention.

The “photodetector device” used should have a high enough sensitivity toenable the imaging of faint light from within a mammal in a reasonableamount of time, and to use the signal from such a device to construct animage.

In cases where it is possible to use light-generating moieties which areextremely bright, and/or to detect light-generating fusion proteinslocalized near the surface of the subject or animal being imaged, a pairof “night-vision” goggles or a standard high-sensitivity video camera,such as a Silicon Intensified Tube (SIT) camera (e.g. HammamatsuPhotonic Systems, Bridgewater, N.J.), may be used. More typically,however, a more sensitive method of light detection is required.

In extremely low light levels the photon flux per unit area becomes solow that the scene being imaged no longer appears continuous. Instead,it is represented by individual photons which are both temporally andspatially distinct form one another. Viewed on a monitor, such an imageappears as scintillating points of light, each representing a singledetected photon. By accumulating these detected photons in a digitalimage processor over time, an image can be acquired and constructed. Incontrast to conventional cameras where the signal at each image point isassigned an intensity value, in photon counting imaging the amplitude ofthe signal carries no significance. The objective is to simply detectthe presence of a signal (photon) and to count the occurrence of thesignal with respect to its position over time.

At least two types of photodetector devices, described below, can detectindividual photons and generate a signal which can be analyzed by animage processor. Reduced-Noise Photodetection Devices achievesensitivity by reducing the background noise in the photon detector, asopposed to amplifying the photon signal. Noise is reduced primarily bycooling the detector array. The devices include charge coupled device(CCD) cameras referred to as “backthinned”, cooled CCD cameras. In themore sensitive instruments, the cooling is achieved using, for example,liquid nitrogen, which brings the temperature of the CCD array toapproximately −120° C. “Backthinned” refers to an ultra-thin backplatethat reduces the path length that a photon follows to be detected,thereby increasing the quantum efficiency. A particularly sensitivebackthinned cryogenic CCD camera is the “TECH 512”, a series 200 cameraavailable from Photometries, Ltd. (Tucson, Ariz.).

“Photon amplification devices” amplify photons before they hit thedetection screen. This class includes CCD cameras with intensifiers,such as microchannel intensifiers. A microchannel intensifier typicallycontains a metal array of channels perpendicular to and co-extensivewith the detection screen of the camera. The microchannel array isplaced between the sample, subject, or animal to be imaged, and thecamera. Most of the photons entering the channels of the array contact aside of a channel before exiting. A voltage applied across the arrayresults in the release of many electrons from each photon collision. Theelectrons from such a collision exit their channel of origin in a“shotgun” pattern, and are detected by the camera.

Even greater sensitivity can be achieved by placing intensifyingmicrochannel arrays in series, so that electrons generated in the firststage in turn result in an amplified signal of electrons at the secondstage. Increases in sensitivity, however, are achieved at the expense ofspatial resolution, which decreases with each additional stage ofamplification. An exemplary microchannel intensifier-based single-photondetection device is the C2400 series, available from Hamamatsu.

Image Processors process signals generated by photodetector deviceswhich count photons in order to construct an image which can be, forexample, displayed on a monitor or printed on a video printer. Suchimage processors are typically sold as part of systems which include thesensitive photon-counting cameras described above, and accordingly, areavailable from the same sources. The image processors are usuallyconnected to a personal computer, such as an IBM-compatible PC or anApple Macintosh (Apple Computer, Cupertino, Calif,), which may or maynot be included as part of a purchased imaging system. Once the imagesare in the form of digital files, they can be manipulated by a varietyof image processing programs (such as “ADOBE PHOTOSHOP”, Adobe Systems,Adobe Systems, Mt. View, Calif.) and printed.

The Detection Field Of The Device is defined as the area from whichconsistent measurements of photon emission can be obtained. In the caseof a camera using an optical lens, the detection field is simply thefield of view accorded to the camera by the lens. Similarly, if thephotodetector device is a pair of “night vision” goggles, the detectionfield is the field of view of the goggles.

Alternatively, the detection field may be a surface defined by the endsof fiber-optic cables arranged in a tightly-packed array. The array isconstructed to maximize the area covered by the ends of the cables, asopposed to void space between cables, and placed in close proximity tothe subject. For instance, a clear material such as plexiglass can beplaced adjacent the subject, and the array fastened adjacent the clearmaterial, opposite from the subject.

The fiber-optic cable ends opposite the array can be connected directlyto the detection or intensifying device, such as the input end of amicrochannel intensifier, eliminating the need for a lens. An advantageof this method is that scattering and/or loss of photons is reduced byeliminating a large part of the air space between the subject and thedetector, and/or by eliminating the lens. Even a high-transmission lenstransmits only a fraction of the light reaching the front lens element.

With higher-intensity LGPs, photodiode arrays may be used to measurephoton emission. A photodiode array can be incorporated into arelatively flexible sheet, enabling the practitioner to partially “wrap”the array around the subject. This approach also minimizes photon loss,and in addition, provides a means of obtaining three-dimensional imagesof the bioluminescence. Other approaches may be used to generatethree-dimensional images, including multiple detectors placed around thesubject or a scanning detector or detectors.

It will be understood that the entire animal or subject need notnecessarily be in the detection field of the photodetection device. Forexample, if one is measuring a light-emitting conjugate known to belocalized in a particular region of the subject, only light from thatregion, and a sufficient surrounding “dark” zone, need be measured toobtain the desired information.

Immobilizing the Subject.

In those cases where it is desired to generate a two dimensional orthree-dimensional image of the subject, the subject may be immobilizedin the detection field of the photodetection devices during the periodthat photon emission is being measured. If the signal is sufficientlybright that an image can be constructed from photon emission measured inless than about 20 milliseconds, and the subject is not particularlyagitated, no special immobilization precautions may be required, exceptto insure that the subject is in the field of the detection device atthe start of the measuring period.

If, on the other hand, the photon emission measurement takes longer thanabout 20 msec, and the subject is agitated, precautions to insureimmobilization of the subject during photon emission measurement,commensurate with the degree of agitation of the subject, need to beconsidered to preserve the spatial information in the constructed image.For example, in a case where the subject is a person and photon emissionmeasurement time is on the order of a few seconds, the subject maysimply be asked to remain as still as possible during photon emissionmeasurement (imaging). On the other hand, if the subject is an animal,such as a mouse, the subject can be immobilized using, for example, ananesthetic or a mechanical restraining device.

In cases where it is desired to measure only the total amount of lightemanating from a subject or animal, the subject does not necessarilyneed to be immobilized, even for long periods of photon emissionmeasurements. All that is required is that the subject be confined tothe detection field of the photodetector during imaging. It will beappreciated, however, that immobilizing the subject during suchmeasuring may improve the consistency of results obtained, because thethickness of tissue through which detected photons pass will be moreuniform from animal to animal.

Further Considerations During Imaging

The visualization of fluorescent light-generating moieties requires anexcitation light source, as well as a photodetector. Furthermore, itwill be understood that the excitation light source is turned on duringthe measuring of photon emission from the light-generating moiety.

Appropriate selection of a fluorophore, placement of the light sourceand selection and placement of filters, all of which facilitate theconstruction of an informative image, are discussed above, in thesection on fluorescent light-generating moieties.

High-Resolution Imaging.

Photon scattering by tissue limits the resolution that can be obtainedby imaging LGMs through a measurement of total photon emission. It willbe understood that the present invention also includes embodiments inwhich the light-generation of LGMs is synchronized to an external sourcewhich can be focused at selected points within the subject, but whichdoes not scatter significantly in tissue, allowing the construction ofhigher-resolution images. For example, a focused ultrasound signal canbe used to scan, in three dimensions, the subject being imaged.Light-generation from areas which are in the focal point of theultrasound can be resolved from other photon emission by acharacteristic oscillation imparted to the light by the ultrasound.

Constructing an Image of Photon Emission.

In cases where, due to an exceptionally bright light-generating moietyand/or localization of light-generating fusion proteins near the surfaceof the subject, a pair of “night-vision” goggles or a high sensitivityvideo camera was used to obtain an image, the image is simply viewed ordisplayed on a video monitor. If desired, the signal from a video cameracan be diverted through an image processor, which can store individualvideo frames in memory for analysis or printing, and/or can digitize theimages for analysis and printing on a computer.

Alternatively, if a photon counting approach is used, the measurement ofphoton emission generates an array of numbers, representing the numberof photons detected at each pixel location, in the image processor.These numbers are used to generate an image, typically by normalizingthe photon counts (either to a fixed, pre-selected value, or to themaximum number detected in any pixel) and converting the normalizednumber to a brightness (greyscale) or to a color (pseudocolor) that isdisplayed on a monitor. In a pseudocolor representation, typical colorassignments are as follows. Pixels with zero photon counts are assignedblack, low counts blue, and increasing counts colors of increasingwavelength, on up to red for the highest photon count values. Thelocation of colors on the monitor represents the distribution of photonemission, and, accordingly, the location of light-generating fusionproteins.

In order to provide a frame of reference for the conjugates, a greyscaleimage of the (still immobilized) subject from which photon emission wasmeasured is typically constructed. Such an image may be constructed, forexample, by opening a door to the imaging chamber, or box, in dim roomlight, and measuring reflected photons (typically for a fraction of thetime it takes to measure photon emission). The greyscale image may beconstructed either before measuring photon emission, or after. The imageof photon emission is typically superimposed on the greyscale image toproduce a composite image of photon emission in relation to the subject.

If it is desired to follow the localization and/or the signal from alight-emitting conjugate over time, for example, to record the effectsof a treatment on the distribution and/or localization of a selectedbiocompatible moiety, the measurement of photon emission, or imaging canbe repeated at selected time intervals to construct a series of images.The intervals can be as short as minutes, or as long as days or weeks.

Analysis of Photon Emission Images

Images generated by methods and/or using compositions of the presentinvention may be analyzed by a variety of methods. They range from asimple visual examination, mental evaluation and/or printing of ahardcopy, to sophisticated digital image analysis. Interpretation of theinformation obtained from an analysis depends on the phenomenon underobservation and the entity being used.

Applications: Localization of Tumor Cells

The growth and metastatic spread of tumors in a subject may be monitoredusing methods and compositions of the present invention. In particular,in cases where an individual is diagnosed with a primary tumor, LGPsdirected against the cells of the tumor can be used to both define theboundaries of the tumor, and to determine whether cells from the primarytumor mass have migrated and colonized distal sites. For example, LGPs,such as liposomes containing antibodies directed against tumor antigensand loaded with LGPs, can be administered to a subject, allowed to bindto tumor cells in the subject, imaged, and the areas of photon emissioncan be correlated with areas of tumor cells.

In a related aspect, images utilizing tumor-localizing LGPs, such asthose described above, may be generated at selected time intervals tomonitor tumor growth, progression and metastasis in a subject over time.Such monitoring may be useful to record results of anti-tumor therapy,or as part of a screen of putative therapeutic compounds useful ininhibiting tumor growth or metastasis.

In the practice of the invention, the tissue and the light-generatingfusion protein can be contacted in vitro, such as where one or morebiological samples (e.g., blood, serum, cells, tissue) are arrayed on asubstrate under tissue culture conditions known by those in the art topreserve the viability of the tissue and then the fusion protein isadded to the tissue culture. In a preferred embodiment of the methods ofthe invention the tissue is mammalian tissue, in particular humantissue.

The methods of the invention can also be practiced in vivo wherein thebiological sample and the light-generating fusion protein are contactedby administration of the fusion protein (or a vector encoding the same)to a subject suspected of containing ischemic tissue under conditions toallow detection of the fusion protein in ischemic tissue present in thesubject. The invention thus also pertains to the field of predictivemedicine in which diagnostic assays, prognostic assays,pharmacogenomics, and monitoring clinical trials are used for prognostic(predictive) purposes to thereby treat an individual prophylactically.Accordingly, one aspect of the invention relates to diagnostic assaysfor determining the presence of ischemic tissue in a biological sampleto thereby determine whether an individual is afflicted with a diseaseor disorder, or is at risk of developing a disorder, associated withhypoxic conditions. The invention also provides for prognostic (orpredictive) assays for determining whether an individual is at risk ofdeveloping conditions arising from ischemia.

Referring now to the Drawings, FIG. 1A is a schematic representation ofdifferent fusion proteins of the present invention. “T” indicates theenzyme target site on the fusion protein, “E” indicates the enzyme, “X”indicates the modification site on the fusion protein, and “R” indicatesthe reporter domain of the fusion protein. The modification at “X” by“E”results in the fusion protein becoming active or inactive “On/Off”.FIG. 1B shows an embodiment wherein the “T” and the “X” are separatedomains and are in cis on the fusion protein. FIG. 1C shows anembodiment of the invention wherein the “T” and the “X” of the fusionprotein are congruent within a domain of the fusion protein. FIG. 1Dshows an embodiment of the invention wherein the “T” is on a proteinassociated with the fusion protein containing the “X”.

FIG. 2 is a schematic representation of different fusion proteins of thepresent invention. FIG. 2A is a schematic representation of a fusionprotein and associated protein of the present invention in which afusion protein and an associated protein “A” contains a known homo- orhetero-dimerization domain “HD” corresponding to the enzyme target siteof an enzyme, where binding of the enzyme-associated “A” to the HD onthe fusion protein results in modification of the fusion protein at “X”.FIG. 2B is a related aspect of the invention in which a binding domainof a protein “A” containing an enzyme target site “T” interacts with abinding domain “B” of a fusion protein, which results in enzyme “E”modifying the reporter domain “R” of fusion protein “B” at modificationsite “X” such that the fusion protein does or does not emit light orcause light to be emitted.

FIG. 3 shows pVHL binding to a modified form of HIF. (A) showspVHL-defective renal carcinoma cells treated with increasing amounts ofdesferoxamine (2, 10, 100, 1000 μm) or cobalt choloride (2, 10, 100,1000 μm) and immunoprecipitated with control (lane 1) or anti HIF2αantibody. Bound proteins were detected by anti-HIF2α immunoblot (IB) orby farwestern (FW) analysis with purified pVHL/elongin B/elongin C (VBC)complexes. (B) shows VBC farwestern and anti-HIF2α immunoblot analysisof ts20 cells grown at the restrictive temperature under hypoxic ornormoxic conditions. (C and D) depict GST-HIF1α(530-652), containing theoxygen-dependent degradation domain (ODD), produced in E. coli,recovered on glutathione Sepharose, and incubated with rabbitreticulocyte lysate for 90 min at 30° C. In (D, lane 3), thereticulocyte lysate was first heat inactivated for 20 min. Followingstringent washes the GST-ODD protein was subjected to VBC farwestern andanti-GST immunoblot analysis.

FIG. 4 shows pVHL binding to a HIF1α-derived peptide if Leu562 andPro564 are intact. (A) shows binding of the indicated ³⁵S-labeledGal4-HIF1α fusion proteins to immobilized GST-pVHL, elongin B, elongin Ccomplexes. (B) shows binding of ³⁵S-labeled pVHL to biotinylated HIF1α(556-575) peptides with the indicated substitutions of residues 561-568.‘+’ indicates preincubation of peptide with unprogrammed reticulocytelysate prior to addition of pVHL. (C and D) depict ³⁵S-labeled wild-type(WT), Pro564Ala, and Leu562Ala full-length HA-HIF1α (panel C) andGa14-HA-HIF1α(530-652) (panel D) proteins immunoprecipitated withanti-HA antibody or captured with immobilized GST-VBC complexes.WG=wheat germ extract ; Retic=rabbit reticulocyte lysate.

FIG. 5 shows ubiquitination and degradation of HIF linked to Leu562 andPro 564. (A) depicts in vitro ubiquitination of ³⁵S-labeled wild-type,Leu562A, and Pro564A Ga14-HA-HIF1α(530-652) in the presence of S100extracts prepared from pVHL-defective renal carcinoma cells stablytransfected to produce wild-type pVHL or with empty vector. (B) shows invitro degradation of ³⁵S-labeled wild-type, Leu562Ala, and Pro564Ala inxenopus egg extracts. (C) is an anti-HA immunoblot analysis of COS7cells transiently transfected with 1.5 or 3.5 μg of plasmids encodingwild-type or P564A HA-HIFα in the absence or presence of desferoxamine.

FIG. 6 depicts proline hydroxylation linked to pVHL-binding. (A) is aMALDI-TOF analysis of wild-type, Pro564Ala, and Leu562Ala biotinylatedHIF (556-575) peptides following incubation with rabbit reticulocytelysate. (B) shows Gal4-HA-HIF (555-575) translated in vitro in thepresence of 3H-Proline with rabbit reticulocyte lysate or wheat germextract and gel-band purified. Following acid hydrolysis proline andhydroxyproline were separated using thin layer chromatography. Thedashed circle indicates positions of ninhydrin stained proline andhydroxyproline markers.

FIG. 7 illustrates that pVHL specifically recognizes HIF1α withhydroxylated proline 564. (A and B) shows binding of ³⁵S-labeled pVHL tobiotinylated HIFα (556-575) peptides with the indicated substitutions ofresidues 561-568. (C) shows ts20 cells stably transfected to produceHA-pVHL grown at restrictive (lane 1) or permissive (lanes 2-6)temperature and immunoprecipitated anti-HIF1α (lane 1 and 2) or anti-HAantibody (lanes 3-7). Bound proteins were eluted by boiling in samplebuffer (lane 1 and 2) or treatment with the indicated peptides and thenimmunoblotted with anti-HIF1α antibody. (D) shows pVHL-defective renalcarcinoma cells stably transfected to produce wild-type pVHL (WT8) orwith empty vector (RC3) metabolically labeled with ³⁵S methionine,lysed, and incubated with immobilized biotinylated HIF1α (556-575)peptides with the indicated substitutions of residue 564. Specificallybound proteins were detected by autoradiography.

FIG. 8 illustrates the production of TETr-cyclins A and E. (A) is aschematic of TETr-cyclin fusion protein. (B) shows production ofTETr-cyclin A and TETr-cyclin E. Cells transfected to produce theindicated cyclin A or cyclin E proteins were lysed and immunoblotted(IB) with the indicated antibodies. (C) shows phosphorylation of pRB byTETr-cyclins A and E in SAOS-2 cells. pRB-defective SAOS-2 cellstransfected so as to produce HA-tagged pRB along with the indicatedcyclins were lysed and immunoblotted with anti-HA antibody. The % oftransfected cells in G1 and S phase was determined by fluororescenceactivated cell sorting (FACS).

FIG. 9 shows DNA bound cyclins A and E differentially affectingtranscription. (A) is a schematic of reporter plasmid (pUHC 13-3) thatcontains seven TETracycline operator sequences (TETo) upstream of aminimal CMV promoter that includes a TATA box. (B, C) show U20S cellswere cotransfected with plasmids encoding the indicated TETr fusionproteins along with the pUHC 13-3 reporter plasmid and a plasmidencoding β-galactosidase. Numbers shown at the bottom of the graphindicate the amount of TETr plasmid (in μg). 48 hours later luciferaseactivity, normalized for β-galactosidase, was determined. Foldrepression is the corrected luciferase value for TETr alone divided bythe corrected luciferase values for the indicated TETr fusion proteins.Fold activation represents the corrected luciferase values for theindicated TETr fusion proteins divided by the corrected luciferase valuefor TETr alone.

FIG. 10 illustrates transcriptional regulation by cyclins A and Edependent upon DNA binding. (A) shows U2OS cells cotransfected withplasmids encoding the indicated TETr fusion proteins along with the pUHC13-3 reporter plasmid and a plasmid encoding β-galactosidase. 24 hourslater doxycycline was added to a final concentration of 2 μg/ml whereindicated by a ‘+’. 24 hours later, luciferase activity, corrected forβ-galactosidase activity, was determined and expressed as foldrepression or activation relative to cells producing TETr alone. (B)shows U2OS cells cotransfected with plasmids encoding the indicatedcyclins along with the pUHC 13-3 reporter plasmid and a plasmid encodingβ-galactosidase. Fold repression and activation was determined as in(A). (C) shows U2OS cells cotransfected with plasmids encoding TETr orTETr-cyclin E, along with a minimal HSV-TK promoter reporter plasmidcontaining the indicated number of TETo binding sites and a plasmidencoding β-galactosidase. Doxycycline was added as in (A).

FIG. 11 illustrates that cyclin box is required for transcriptionalrepression by DNA bound cyclin A. (A) U2OS cells were cotransfected withplasmids encoding the indicated TETr-cyclin A variants along with pUHC13-3 reporter plasmid and a plasmid encoding β-galactosidase. Cellextracts were prepared and luciferase activity, corrected forβ-galactosidase activity, was expressed as fold repression relative tocells producing TETr alone. (B) The indicated TETr-cyclin A variantswere translated in vitro in the presence of ³⁵S-methionine and incubatedwith GST-cdk2 and glutathione Sepharose. Specifically bound proteinswere resolved by SDS-polyacrylamide gel electrophoresis and detected byautoradiography. In parallel, 20% of the input proteins were resolved bySDS-polyacrylamide gel electrophoresis and detected by autoradiography.(c) pRB defective SAOS-2 cells transfected so as to produce HA-taggedpRB along with the indicated TETr-cyclin A variants were lysed andimmunoblotted with anti-HA antibody.

FIG. 12 shows transcriptional activation by cyclin E linked to itsability to bind to cdk2 and interact with substrates. (A) U2OS cellswere cotransfected with plasmids encoding the indicated TETr-cyclin Evariants along with the pUHC 13-3 reporter plasmid and a plasmidencoding β-galactosidase. Cell extracts were prepared, and luciferaseactivity, corrected for β-galactosidase activity, was expressed as foldactivation relative to cells producing TETr alone. (B) The indicatedTETr-cyclin E variants were translated in vitro in the presence of³⁵S-methionine and incubated with GST-cdk2 and glutathione Sepharose.Specifically bound proteins were resolved by SDS-polyacrylamide gelelectrophoresis and detected by autoradiography. In parallel, 20% of theinput proteins were resolved by SDS-polyacrylamide gel electrophoresisand detected by autoradiography. (c) pRB defective SAOS-2 cellstransfected so as to produce HA-tagged pRB along with the indicatedTETr-cyclin E variants were lysed and immunoblotted with anti-HAantibody.

FIG. 13 shows transcriptional activation by DNA bound cyclin E dependenton cdk2 catalytic activity. (A, B) U2OS cells were transientlycotransfected with plasmids encoding TETr-cyclin A or E and, whereindicated, increasing amounts of a plasmid encoding a dominant-negative(dn) form of cdk2. Cell extracts were prepared and luciferase activity,corrected for β-galactosidase activity, was determined. Correctedluciferase values were expressed as fold repression (A) or activation(B) relative to TETr alone. (C) U2OS cells were transiently transfectedwith plasmids encoding TETr-cdk2 or TETr-cdk2 (N132A) and a plasmidencoding either cyclin A or E. Cell extracts were prepared andluciferase activity, corrected for β-galactosidase activity, wasdetermined. Corrected luciferase values were expressed as foldactivation relative to TETr alone.

FIG. 14 shows transcriptional effects mediated by cell-cycle dependentchanges in endogenous cyclins E and A. (A, B) 3T3 cells stablytransfected with a luciferase reporter plasmid containing 7 TETo sites(pUHC13-3) and a plasmid encoding TETr-cdk2 were serum-starved for 72hours and subsequently re-fed with serum. At various timepointsthereafter aliquots of cells were removed and either lysed forimmunoblot analysis with the indicated antibodies or analyzed for DNAcontent by propidium iodide staining followed by fluorescence activatedcell sorting (FACS). (C) 3T3 cells stably transfected with a luciferasereporter plasmid containing 7 TETo sites (pUHC13-3) in the absence (opencircles) or presence of a plasmid encoding TETr-cdk2 (closed squares) orTETr-cdk2 (N132A) (open squares) were serum-starved for 72 hours andthen re-fed with serum in the presence or absence of doxycycline. Atvarious timepoints thereafter luciferase assays were performed. Tocorrect for general effects due to serum, the luciferase values at eachtimepoint in the absence of doxycycline were corrected by subtractingthe luciferase assay obtained in the presence of doxycycline. Aftercorrection, the luciferase values for the two cell populations wereexpressed relative to the corresponding luciferase values obtained attime 0. In parallel, the cells producing TETr-cdk2 were lysed andimmunoprecipitated with anti-cyclin A or anti-cyclin E antibodies. Theimmunoprecipitates were then used to phosphorylate Histone H1 in vitro.

Methods of Treating Hypoxia or Ischemia Related Tissue Damage andModulating Angiogenesis or Vascularization.

Tissue ischemia is a major cause of morbidity and mortality. Inprinciple, drugs that stabilize HIF may augment angiogenesis and theadaptation of hypoxia. The activation of HIF by hypoxia is complex andinvolves protein stability, nuclear localization, DNA binding capabilityand transcriptional activation function. The discovery that prolinehydroxlaytion governs HIF turnover in the presence of oxygen willfacilitate the dissection of the mechanism underlying the variousaspects of HIF regulation.

The invention also provides various methods of treating, i.e., reducing,preventing or delaying the onset of HIF-1 related disorders, modulatingangiogenesis or vascularization. Examples of HIF-1 mediated disordersinclude chronic and acute hypoxia or ischemia related disorders such astissue damage and scarring. Acute hypoxia or ischemia related disordersinclude for example myocardial infarction, stroke, cancer and diabetessuch as tissue damage and scarring. Chronic hypoxia or ischemia relateddisorders include for example, deep vein thrombosis, pulmonary embolusand renal failure.

In one aspect the invention the invention provides methods of treatingor preventing a hypoxic or ischemic related disorder or modulatingangiogenesis or vascularization by administering to a subject a compoundthat decreases prolyl hydroxylase, expression or activity. Examples ofprolyl hydroxylase include human Egl-9 or homologs. (Epstein, et al Cell107:43-54, 2001) The compound can be a prolyl hydroxylase antibody, anucleic acid that decreases the expression of a nucleic acid thatencodes a prolyl hydroxylase polypeptides such as a prolyl hydroxylaseanti-sense nucleic acid or a compound identified by any of the methodsof the invention. Preferably, the half life of HIF in the subject isincreased in the presence of the compound as compared to the absence ofthe compound.

In a further aspect the invention includes a method of treating cancerin a subject by administering to the subject a compound that increasesprolyl hydroxylase expression or activity. Preferably, the compound is acompound that has been identified by the methods of the invention.

In another aspect the invention provides a method for treating orpreventing a hypoxia or ischemic related disorder in a subject byadministering to a subject a compound that which modulates prolylhydroxylation of HIF.

In still a further aspect the invention provides a method for treatingor preventing a HIF related disorder by administering to a subject acompound that which modulates prolyl hydroxylation of HIF such that theHIF related disorder is prevented reversed of stabilized.

In yet another aspect the invention provides a method for regulating HIFturnover in a subject by administering to a subject a compound thatwhich modulates prolyl hydroxylation.

By “modulates” is meant to increase or decrease the prolyl hydroxylationof HIF. Compounds that inhibits prolyl hydroxylation include severalsmall molecule proline hydroxylase inhibitors which have been developedas antifibrotic agents.

The subject is preferably a mammal. The mammal can be, e.g., a human,non-human primate, mouse, rat, dog, cat, horse, or cow. In variousaspects the subjects include patients with coronary, cerebral, orperipheral arterial disease and patients with one or more non-healingwounds.

EXAMPLES Example 1. Characterization of Hypoxia-Responsive Polypeptides

In order to demonstrate the efficacy of the First embodiment of theinvention, e.g. employing a hypoxia-responsive LGP, the interaction ofpVHL and HIF was examined. A HIF1α polypeptide that is sufficient tobind pVHL is disclosed herein. pVHL binds directly to a region of HIF1αcalled the oxygen-dependent degradation domain (ODD). pVHL recognizesHIF produced in rabbit reticulocyte lysate but not HIF produced in wheatgerm extracts or in E. Coli. Furthermore, wheat germ or E. Coli-derivedHIF binds to pVHL following preincubation with a human, rabbit, orxenopus cell extracts at 37° C. For example, glutathioneS-transferase-ODD fusion proteins produced in E. Coli were notrecognized by VBC in farwestern assays (FIG. 3C). These proteins wererecognized, however, after pre-incubation with a rabbit reticulocytelysate. Similar results were obtained with GST-ODD fusion proteins ofvarious sizes, thus excluding the possibility that the farwestern blotsignal represents a spurious interaction between VBC and areticulocyte-derived protein. VBC did not recognize GST-ODD fusionproteins incubated with a heat-inactivated reticulocyte lysate (FIG.3D). Gal4-HIF fusion proteins containing HIF residues 555-575 boundspecifically to immobilized GST-VHL, elongin B, elongin C complexes(FIG. 4A). Coupled in vitro transcription/translation of ³⁵S-labeledproteins was conducted according to the manufacturer's instructions(TNT, Promega). Also, a biotinylated peptide corresponding to HIFresidues 556-575 bound to pVHL following pre-incubation withreticulocyte lysate (FIG. 4B). For peptide binding studies, 1 μg ofbiotinylated peptide was bound to 30 μl of monomeric avidin Sepharose(Pierce). Where indicated, the peptide was pre-incubated with 50 μl ofrabbit reticulocyte lysate for 90 min at 30° C. The Sepharose was thenwashed 3 times with NETN (20 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA,0.5% non-idet P40) and used in binding reactions containing 4 μl³⁵S-HA-pVHLin 500 μl of EBC or 500 μl ³⁵S-radiolabeled cell extract(equivalent to cells from a subconfluent 100 mm dish). Following 1 hourincubation at 4° C. with rocking the Sepharose was washed 4 times withNETN. Bound proteins were eluted by boiling in SDS-containing samplebuffer and detected by autoradiography. This region of HIF contains ahighly conserved 8 mer (MLAPYIPM) which, when mutated to 8 consecutivealanines, leads to HIF stabilization in cells. An alanine scan of thisregion showed that Leu562 and Pro564 were essential for specific bindingto pVHL in this assay (FIG. 4B). In contrast, mutation of the onepotential phosphoacceptor in this peptide, Tyr565, did not affect pVHLbinding, in keeping with an earlier study in which a Tyr565Phe mutationdid not affect HIF stability. In addition, the binding of pVHL toGST-ODD in the assays described above was unaffected by phosphatasetreatment.

Binding of pVHL to HIF1α is critically dependent upon residues Leu562 orPro564 of HIF1α. Importantly, mutation of either Leu562 or Pro564 in thecontext of full-length HIF1α or a Gal4-ODD fusion protein also led to aloss of pVHL binding activity (FIG. 4C and 4D, respectively). Gal4-ODDmade with reticulocyte lysate contained an electrophoretically distinctband compared with Gal4-ODD made with wheat germ extract (FIG. 4D). Thiselectrophoretically distinct protein bound to VBC and was undetectableamong the Leu562Ala and Pro564Ala translation products. The isoelectricpoints of the two arrowed bands in FIG. 4D were identical following 2-Dgel electrophoresis indicating that the putative modification did notinvolve a change in protein charge.

Modification of HIFαby pVHL following binding is also criticallydependent upon residues Leu562 or Pro564 of HIFlα. Gal4-HIF fusionproteins with the Leu562Ala mutation or Pro564Ala mutation displayeddiminished pVHL-dependent polyubiquitination in vitro relative to thecorresponding wild-type protein (FIG. 5A). Qualitatively similar resultswere obtained with the corresponding full-length HIF1α species, althoughthis assay is less robust than with the Gal4-ODD fusion proteins.Likewise, HIF1α Pro564Ala and HIF1α Leu562Ala were far more stable thanwild-type HIF1α in in vitro degradation assays performed with xenopusextracts (FIG. 5B). Xenopus egg extracts were made as is well known inthe art and stored frozen until use. Degradation reactions contained 8μl of egg extract, 0.1 μl of 100 mg/ml cyclohexamide, 0.25 μl of energyregeneration mix, 0.25 μl of bovine ubiquitin, and 0.4 μl of³⁵S-radiolabeled HIF and were carried out at room temperature. At theindicated timepoints 1 μl aliquots were removed and placed in samplebuffer. Samples were resolved on 5-15% gradient gels and analyzed byautoradiography. Biotinylated HIF (556-575) peptides were incubated withrabbit reticulocyte lysate, washed, eluted with free biotin, andanalyzed by mass spectrometry. 1 μl of HPLC-purified peptide was boundto 30 μl monomeric avidin Sepharose and incubated with 100 μl of rabbitreticulocyte lysate at room temperature for 1 hour with tumbling.Following brief centrifugation the reticulocyte lysate was removed,fresh reticulocyte lysate was added, and the cycle was repeated 6 times.The Sepharose was then washed 4 times with NETN and once with PBS. Themodified peptide was eluted in 50 μl of 20 mM ammonium acetate [pH 7.0],2 mM biotin. In these experiments Met561 and Met568 were converted toalanine to prevent spurious oxidation of the methionine residues duringanalysis. This double alanine substitution, like the correspondingsingle substitutions, did not affect pVHL binding. Treatment of the HIF(556-575) peptide with rabbit reticulocyte lysate led to the appearanceof a second peak in MALDI-TOF assays representing an increase inmolecular weight of 16 (FIG. 6A). This peak was not detectable prior toincubation with reticulocyte lysate and was not detected in thecorresponding reticulocyte-treated Leu562Ala and Pro564Ala peptides(FIG. 6A). Postsource decay analysis using the same instrument placedthe addition of +16 at Pro 564. Similarly, electrospray ion trap massspectrometry/mass spectrometry (MS/MS) analysis was consistent withaddition of +16 at Pro 564 and excluded such a modification of Leu 562.Finally, MS/MS analysis of the reticulocyte-treated HIF (556-575)peptide produced a pattern of ions that was identical to the patternobtained with a HIF (556-575) peptide which was synthesized to containhydroxyproline at position 564. Next, Gal4-HIF (555-575) was translatedin vitro in the presence of ³H-Proline using rabbit reticulocyte lysateor wheat germ extract, gel-band purified, and subjected to acidhydrolysis and thin layer chromatography. 2 ml of ³H-P-labeled Gal4-HIF(555-575) in vitro translate was immunoprecipitated with 50 μg ofanti-HA antibody (12CA5, Roche), resolved on a 12% SDS-polyacrylamidegel, and transferred to a PVDF membrane. Ga14-HIF (555-575) wasvisualized by autoradiography and the corresponding region of PVDF wasexcised, hydrolyzed by incubation in 100 μl of 10 N HC1 at 105° C. for 3hours. Samples were evaporated to dryness, resuspended in 20 μl H₂Ocontaining 10 μg of unlabeled proline and 4-OH proline (Sigma), andresolved by 2-D thin layer chromatography using phenol-distilled H₂O inthe first dimension and N-butanol-acetic acid-H₂O in the second.Following visualization of standards with ninhydrin radiolabeled prolinewas detected by autoradiography. Gal4-HIF produced in rabbitreticulocyte lysate, but not in wheat germ, contained hydroxyproline(FIG. 6B).

pVHL specifically recognizes a proline hydroxylated determinant. The HIF(556-575) peptide containing hydroxyproline at position 564 bound topVHL with or without pretreatment with reticulocyte lysate (FIG. 7A).The mass spectrometry analysis of the Leu562Ala peptide showed thatLeu562 was required for HIF modification (FIG. 6A). Indeed, pVHL boundto a HIF (556-575) peptide with the Leu562Ala substition andhydroxyproline at residue 564 (FIG. 7B). This suggests that the primaryrole of Leu562 is to allow for the hydroxylation of Pro564. Twoapproaches were used to demonstrate that hydroxylated HIF peptide couldinteract with cell-derived pVHL complexes. Ts20 cells were engineered toproduce HA-tagged pVHL which carry a temperature-sensitive mutation inthe El-ubiquitin activating enzyme. ts20 cells were transfected withpIRES-HA-VHL, pIRES-HA-VHL (Y98H), or pIRES-Neo (Invitrogen) andselected in the presence of 1 mg/ml G418. Individual G418-resistantcolonies were isolated using cloning cylinders and expanded. Cellsproducing HA-VHL or HA-VHL (Y98H) were identified by anti-HA immunoblotanalysis. HIF coimmunoprecipitated with HA-pVHL at the restrictivetemperature. HIF bound to pVHL in this way could be eluted by thehydroxylated HIF (556-575) peptide but not the unmodified peptide (FIG.7C). For the tests shown in FIG. 7C, ts20 cells were grown at therestrictive or permissive temperature for 14 hour, methionine-starvedfor 90 min, and then grown in the methionine-free media supplementedwith ³⁵S-met (500 mCi/ml) for 90 min. Cells were washed once with coldPBS, lysed in EBC, and immunoprecipitated with anti-HA (12CA5; Roche oranti-HIF1α (NB100-105; Novus). Following 5 washes with NETN boundproteins were eluted by boiling in sample buffer or by incubation in 65μl of PBS containing 7 μg of the indicated peptide. Moreover, HIF wasnot eluted by the HIF (556-575) Pro564Ala peptide or by apoly-hydroxyproline peptide (FIG. 7C) Affinity chromatography wasperformed with immobilized peptides and metabolically labeled matchedrenal carcinoma cells which do (WT8) or do not (RC3) produce HA-pVHL.786-O subclones were starved for 1 hour, grown in the methionine-freemedia supplemented with ³⁵S-met (500 mCi/ml) for 3 hr, washed once withice cold PBS, and lysed in EBC. The proline hydroxylated HIF (556-575)peptide specifically bound to pVHL as well as proteins with the expectedelectrophoretic mobilities of the pVHL-associated proteins elongin B,elongin C, and Cu12 (FIG. 7D). The identity of pVHL in this experimentwas confirmed by western blot analysis. Likewise, the binding ofendogenous pVHL to the hydroxylated peptide was detected using 293embryonic kidney cells. A HIF1α mutant in which Proline 564 wasconverted to alanine was stabilized in cells and insensitive to thehypoxia-mimetic desferoxamine (FIG. 5C).

The HIF1α protein is stabilized in hypoxic conditions and functions toinhibit, decrease and/or reverse hypoxia in affected tissues, e.g. in asolid tumor, diabetic retinopathy or ischemic heart tissue, in part bymodulating pro-angiogenic factors. A light-generating fusion proteincontaining a HIF1α moiety will also be stabilized in hypoxic tissue.Destruction of a HIF1α protein or a HIF1α-containing LGP via the pVHLubiquitin pathway occurs, e.g. during normoxia, when a prolylhydroxylase modifies the HIFla protein such that pVHL binds to andmodifies HIF1α. This modification process acts as a dynamic switch toregulate the bioluminescence of the HIF1α-containing light-generatingfusion protein such as the First embodiment of the invention. AHIF1α-containing light-generating fusion protein is useful todynamically image hypoxic tissues, e.g. cancer, in situ, and to screenfor or test the efficacy of hypoxia-modulating compounds.

Example 2

The following example demonstrates the efficacy of light-generatingfusion proteins of the invention for imaging hypoxic tissues.

The Oxygen-Dependent Degradation Domain (ODD) of HIF1α, renders HIF1αunstable in the presence of oxygen. This region is recognized by pVHL.pVHL specifically binds directly to peptidic determinants, correspondingto HIF1α residues 555-575, located within the ODD. At the core of thispeptide there is a conserved proline residue (residue 564) which, in thepresence of oxygen, becomes enzymatically hydroxylated. This residueserves as the signal for pVHL to bind. In sum, in the presence ofoxygen, the HIF peptide becomes hydroxylated and is recognized by pVHL.In the absence of oxygen (hypoxia), the modification does not take placeand pVHL does not bind to HIF.

Construction of ODD-LUC plasmids

In a first set of experiments the ability of a small HIF peptide(555-575) to function in cis to target a foreign protein forpVHL-dependent proteolysis was evaluated. To this end, the HIF cDNAencoding amino acids 555-575 (hereafter ‘ODD’) was PCR amplified witholigonucleotides that introduced convenient restriction sites, digested,and gel-band purified. The PCR fragment was then subcloned, in-frame, 3′of a Firefly luciferase cDNA contained in the pGL3 plasmid (Promega).The resulting luciferase-ODD chimeric cDNA was subcloned into pcDNA3(Invitrogen) to facilitate in vitro translation and expression studiesin mammalian cells. In parallel a pcDNA3 plasmid encoding wild-typeFirefly luciferase and pcDNA3 plasmid encoding wild-type HIF1α was usedas controls.

VBC-GST Pulldown of ODD-Luciferase

To determine whether the ODD-luciferase protein could bind to pVHL,GST-VHL, elongin B, and elongin C (‘GST-VBC’) were produced in E. Coliand recovered as a trimeric complex on glutathione sepharose. Earlierwork showed that pVHL does not fold properly in the absence of elongin Band elongin C. HIF1α, ODD-luciferase, and wild-type luciferase weretranslated in vitro using rabbit reticulocyte lysate (RRL) or wheat germextract (WG) in the presence of ³⁵S methionine (FIG. 15). Aliquots fromthe in vitro translation reactions were added to recombinant VBC-GSTprebound to glutathione sepharose and incubated for 1 hour. Thesepharose was then washed and bound proteins were resolved on a 12% SDSpolyacrylamide gel. The gel was then dried and exposed to film. pVHLbound to reticulocyte-generated HIF1α and ODD-Luc, but not Luc (comparelanes 4, 8 and, 12). Furthermore, pVHL did not bind to wheat germproduced ODD-Luc, as wheat germ lacks the HIF prolyl hydroxylase(compare lanes 6 and 8). Thus the data show that ODD-Luc wasspecifically recognized by pVHL.

Peptide Competition Assay

The observation that pVHL bound to ODD-Luc produced in RRL, but not WG,strongly suggested that the ODD-Luc, like HIF, needed to undergo prolylhydroxylation in order to be recognized by pVHL. To study this further,the GST-VBC binding experiments were repeated in the presence ofsynthetic HIF (555-575) peptides in which Pro 564 was hydroxylated(P-OH) or was not hydroxylated (P). GST-VBC was mixed with either thecontrol (P) peptide or the P-OH peptide in increasing concentrations(from 0-5 μg). Next in vitro translated (in retic lysate) Luc, ODD-Luc,or HIF1α was then added to see if it could efficiently compete off anybound P—OH. HIF and ODD-Luc were able to compete off the P—OH peptideeffectively at even very high concentrations of P—OH. Thus, the resultsobtained with ODD-Luc recapitulated earlier binding studies performedwith authentic HIF. (FIG. 16.)

Luciferase Activity in Cells

In pilot experiments, it was confirmed that ODD-Luc in vitro translateretained luciferase activity in vitro comparable to wild-typeluciferase. To begin to ask whether the ODD-Luc was oxygen sensitive incells, transient transfection assays were performed. In the first set ofexperiments, 100 mm tissue culture plates were seeded with 8×10⁵ HeLacells per plate. Eighteen hours later the cells were transientlytransfected, using lipofectamine (Gibco), with the pcDNA3 plasmidsencoding ODD-Luc or wild-type Luc along with an internal control plasmidencoding Renilla luciferase. Twenty-four hours after transfection thecells were split into 6 well plates and allowed to adhere and grow for8-12 hours. At this point the hypoxia mimetics desferrioxamine (DFO) orcobalt chloride (CoCl2) were added directly to the media at finalconcentrations of 500 mM and 200 mM respectively, in duplicate wells. Inaddition, some wells were not treated so as to serve as controls. 12hours after treatment with DFO and CoCl₂ the cells were lysed withPassive Lysis Buffer (Promega), rocked at room temperature for 20minutes, and assayed for Firefly and Renilla Luciferase according to themanufacturers instructions. Results were obtained in duplicate andaveraged. (FIG. 17).

In the untreated cells the luciferase values for ODD-Luc wereapproximately 30% of the values obtained with wild-type Luc. Note thatHeLa cells have wild-type pVHL and these experiments were conductedusing cells grown in the presence of oxygen. The addition ofhypoxia-mimetics led to a marked increased in ODD-Luc luciferaseactivity, whereas no such induction was detected with wild-typeluciferase. The induced levels of ODD-luciferase were comparable tothose obtained with wild-type luciferase. These results are consistentwith the idea that ODD-Luc is subject to pVHL-dependent proteolysis incells whereas wild-type Luc is not.

Imaging of ODD-Luc in Xenograft Tumors in Nude Mice.

To verify in a mammalian system that the ODD-Luc gene is indeedregulated by hypoxia or hypoxia-mimetics, the following experiment wasconducted. Given the ease of subcutaneously transplanting tumors andtheir ability to grow and vascularize, the ability to image luciferaseactivity of tumors at the subcutaneous level and assess ODD-Luc'sresponse to hypoxia was tested as follows.

Polyclonal Hela cells stably transfected with ODD-Luc and Luc weregenerated. The clones were suspended in PBS and counted. 10⁶ cells perinjection site were administered subcutaneously in duplicate into theflanks of nude mice (FIG. 18). Upon growth of palpable tumors(approximately 3-4 days) the mice were given intraperitoneal injectionsof phenobarbital for anesthesia, injected with a weight-adjusted dose ofluciferin, and whole body imaged with a Xenogen Camera. To ensure thatthe difference in luciferase activity was not simply a function of tumorsize alone, bidimensional tumor measurements were taken and wereapproximately equal.

FIG. 18 shows three different mice injected with ODD-Luc (right) and Luc(left) in duplicate. The sites where ODD-Luc was injected clearly haveattenuated luciferase signal. As such, the data show a real attenuationof luciferase activity, secondary ubiquitination and destruction ofODD-Luc.

Example 3. Light-Generating Fusion Proteins Including a DNA Binding Site

In order to demonstrate the efficacy of the Second embodiment of theinvention, a LGP capable of interacting with nucleic acids in order tokinetically monitor gene transcription in vivo, in vitro or in silico,the impact of cyclins on transcription was examined. As it is useful tomonitor either the induction or repression of transcription, cyclinswith specific affects on transcription were investigated.

Fusion of a DNA-binding motif to a cyclin does not alter cyclin bindingto a cdk or the kinase activity of the cyclin/cdk complex. Mammalianexpression plasmids were generated that encode fusion proteinsconsisting of the TET repressor DNA-binding domain (TETr) (Gossen andBujard, 1992) fused to cyclin A or cyclin E with an intervening flexiblelinker consisting of Gly₄-Ser repeats (FIG. 8A). Both of these plasmidsgave rise to stable proteins of the expected size following transfectioninto mammalian cells (FIG. 8B). TETr-cyclin A and TETr-cyclin E, liketheir unfused counterparts, bound to cdk2 (FIGS. 11 and 12) and couldphosphorylate p107 in vitro. Furthermore, both TETr-cyclin A andTETr-cyclin E promoted pRB phosphorylation and bypassed a pRB-inducedG1/S block when cointroduced with wild-type pRB into pRB-defective tumorcells (FIG. 8C).

Cyclin A and cyclin E dramatically affect transcription. U2OS cells weretransiently transfected with plasmids encoding various TETr fusionproteins and a luciferase reporter plasmid containing 7 TETo bindingsites upstream of a TATA box derived from the CMV promoter (FIG. 9A).TETr binds specifically to TETo sites. As expected, TETr-RB repressedtranscription from this reporter plasmid whereas TETr-E2F1 activated thereporter (FIGS. 9B-C). The basal activity observed with this reporterplasmid presumably reflects the presence of cryptic enhancer sequences.In this and subsequent assays, the TETr domain alone was essentiallyinert. Surprisingly, TETr-cyclin A and TETr-cyclin E both dramaticallyaffected transcription in this assay and did so in opposite ways.TETr-cyclin A decreased transcription approximately 80% (5-foldrepression) whereas TETr-cyclin E increased transcription 10-fold (FIGS.9B-C). Doxycycline prevents the binding of TETr to TETo and completelyblocked the transcriptional effects of TETr-cyclin A and TETr-cyclin E(FIG. 10A). As expected, doxycycline also blocked the transcriptionaleffects of TETr-RB and TETr-E2F1, which were tested in parallel.Furthermore, unfused cyclin A and E had no effects on the TETo-drivenreporter plasmid (FIG. 10B). Experiments were repeated using reporterscontaining 1, 2, 3, or 7 TETo in which the CMV-derived TATA box wasreplaced with a minimal HSV TK promoter (Gossen and Bujard, 1992) (FIG.10C). TETr-cyclin E also activated these reporters indoxycycline-inhibitable manner. The degree of activation observed withthe HSV TK series of reporters was lower than with the CMV TATA-basedreporter, in keeping with earlier results obtained with these reportersand fused TETr to the HSV VP16 transcriptional activation domain (Gossenand Bujard). The low basal level of transcription from these reportersprecluded analysis of repression by cyclin A.

The specific domains of the cyclins that bind to the cdks are criticalfor cyclin-mediated transcriptional regulation. Plasmids encoding TETrfused to various colinear fragments of cyclin A and E were used todetermine which regions of these molecules are required fortranscriptional regulation (FIGS. 11 and 12). All of the resultingfusion proteins were expressed at comparable levels in transienttransfection experiments. Cyclin A (1-310), like wild-type cyclin A,repressed transcription when fused to TETr (FIG. 11A). This fragment ofcyclin A does not bind to cdk2 (FIG. 11B) and cannot direct thephosphorylation of pRB when introduced into cells (FIG. 11C).Conversely, a cyclin A point mutant (E220A) (Schulman et al.,1998) thatmeasurably interacts with cdk2 (FIG. 11B) and directs thephosphorylation of pRB (FIG. 11C) did not repress transcription in theseassays (FIG. 1IA). This mutation maps to the cyclin A cyclin box (FIG.11A). TETr-cyclin A also repressed transcription when tested in p107-/-;p130-/- mouse fibroblasts and cyclin A (1-310) does not bind to eitherp107 or p130. Only those cyclin E mutants that bind to cdk2 (FIG. 12B)and could direct the phosphorylation of pRB (FIG. 12C) scored astranscriptional activators (FIG. 12A). For example, Schulman et al(1998) identified cyclin A residues that are critical for substratebinding and assembly with cdk2. Mutation of analogous residues in cyclinE produced a mutant (cyclin E L134A/Q174A) that likewise failed to bindto cdk2 (FIG. 12B) and failed to phosphorylate pRB (FIG. 12C). Thismutant did not activate transcription (FIG. 12). In keeping with theseresults, a dominant-negative form of cdk2 blocked transcriptionalactivation by cyclin E (FIG. 13B) but had no effect on transcriptionalrepression by cyclin A (FIG. 13A). Similarly, cyclin E, but not cyclinA, activated transcription in concert with a TETr-cdk2 fusion providedthe kinase domain was intact (FIG. 13C). Comparable production ofTETr-cdk2 and kinase-defective TETr-cdk2(N132A) was confirmed byimmunoblot assay. Xenopus cyclin E (Jackson et al.,1995), like its humancounterpart, also activated transcription in these assays (data notshown). This activity was specific as xenopus cyclin E variants withpoint mutations affecting the cyclin box were inert.

The Second embodiment of the invention is useful to dynamically imagetranscription in vivo. For example, cyclin E can activate transcriptionunder physiological conditions. 3T3 cells were transfected with theplasmid containing a selectable marker and TETo reporter plasmid with orwithout a plasmid encoding TETr-cdk2. Following drug selection, thestable transfectants were maintained as polyclonal pools and serumstarved into quiescence. At various timepoints after serum refeeding,cell lysates were prepared and used in immunoblot, in vitro kinase, andluciferase assays (FIG. 14). In parallel, aliquots of the cells wereanalyzed for DNA content by FACS. In this system, S-phase entry began18-20 hours following the addition of serum. As expected, luciferaseactivity increased in the TETr-cdk2 producing cells coincident with anincrease in cyclin E protein levels and cyclin E-associated kinaseactivity (FIGS. 14A-C). No such increase was observed in the cellsproducing equivalent amounts of TETr-cdk2 (N132A) or transfected withthe reporter alone (FIG. 14C and data not shown). Note that the amountof TETr-cdk2 in these cells was less than the amount of endogenous cdk2(FIG. 14B). Thus, the results are unlikely to be an artifact ofoverproduction. Luciferase values declined as cyclin E levels fell andcyclin A levels began to rise.

The Second embodiment of the invention in part relates to LGPscontaining cyclin binding domains. Thus, these LGPs are useful todynamically quantify alterations in transcription of cell-cycleassociated genes, such as oncogenes and tumor suppressors. A LGPcontaining a cyclin-binding moiety can be localized to regionsundergoing cell proliferation, such as a tumor, and can be used toscreen for and determine the efficacy of cell proliferation modulatingcompounds.

MATERIALS AND METHODS Cell Lines and Transfection

U2OS human osteosarcoma cells were grown in Dulbecco's modified Eaglemedia (DMEM) supplemented with 10% heat-inactivated fetalclone (Hyclone)(FC), 100 units/ml penicillin, 100 mg/ml streptomycin, and 2 mML-glutamine (PSG). SAOS-2 human osteosarcoma cells and NIH 3T3 mousefibroblast cells were grown in DMEM supplemented with 10%heat-inactivated fetal bovine serum (FBS) and PSG. NIH 3T3 stablesubclones transfected with the pCMV-neo and pUHC13-3 reporter plasmidalone or with pSG5-TETr-cdk2 or with pSG5-TETr-cdk2(N132A) weremaintained in 0.7 mg/ml of G418. Cells were transfected using2×Bes-buffered saline (2×BBS)/calcium phosphate as described (Chen andOkayama, 1987). Where indicated doxycycline (Sigma) was added 24 hoursafter transfection to a final concentration of 2 μg/ml. Cells weremaintained in doxycycline for an additional 24 hours prior to harvest.

Plasmids

pRcCMV-cdk2 dominant negative (van den Heuvel and Harlow, 1993) was agift of Dr. Ed Harlow; pVL1393-cdk2(N132A) (Xu et al., 1994) was a giftof Dr. Helen Piwnica-Worms; pCD19 (Tedder and Isaacs, 1989) was a giftof Dr. Thomas Tedder and pUHC13-3; ptet1-T81luc, ptet2-T81-luc,ptet3-T81-luc, and ptet7-T81-luc (Gossen and Bujard, 1992) were gifts ofDr Manfred Gossen. pSG5-TETr-E2F1, pSG5-TETr-RB, pSG5-HA-RB (Sellers,1995), pGEX-2TK-cdk2 (Adams et al., 1996) have been describedpreviously. To make pSG5-TETr-cyclin A, a protein phosphatase 1 (PP1)cDNA was first PCR amplified with oligos5′-GCGCTGATCAGGCGGAGGCGGATCAGGAGGAGGAGGATCAGGCGGAGGAGGATCAGGATCCATGTCCGACAGCGAGAA-3′ (SEQ ID NO: 7) and5′-GCGCGAATTCATTTCTTGGCTTTGGCAGA-3′ (SEQ ID NO: 8). The PCR product wascut with Bc1I and EcoRI and subcloned into pSG5-TETr cut with BamHI andEcoRI to make pSG5-TETr-(G1Y₄-Ser)₃-PP1. The cyclin A open reading frame(ORP) was PCR amplified with primers that introduced a 5′ BamHI site anda 3′ EcoRI site and subcloned into pSP72 (Promega) cut with these twoenzymes to make pSP72-cyclin A. The PPI ‘suffer ’frompSG5-TETr-(Gly₄-Ser)₃-PP1 was then excised by digestion with BamHI andEcoRI and replaced with the cyclin A cDNA insert from pSP72-cyclin A. Tomake pSG5-TETr-cyclin E, the cyclin E ORF in pRcCMV-cyclin E was PCRamplified with primers that introduced a 5′ BglII and 3′ EcoRI site. ThePCR product was cut with these two enzymes and ligated into theBamHI-EcoRI backbone of pSG5-TETr-PP1. In parallel, these restrictedcyclin A and cyclin E PCR products were subcloned into pSG5-HA cut withBamHI and EcoRI to make pSG5-HA-cyclin A and pSG5-HA-cyclin E,respectively. Plasmids encoding cyclin A and cyclin E N-terminal andC-terminal deletion mutants were made in an analogous fashion by usingPCR primers that selectively amplified the desired coding regions. Tomake pSG5-TETr-cdk2 and pSG5-TETr-cdk2 (N132A), the cdk2 ORF inpRcCMV-cdk2 and pVL1393-cdk2 (N132A), respectively, were PCR amplifiedwith primers that introduced a 5′ BamHI and 3′ EcoRI site. The PCRproducts were cut with these two enzymes and ligated into theBamHI-EcoRI backbone of pSG5-TETr-PP1. All PCR reactions were performedwith Pfu DNA polymerase and the authenticity of plasmids containing theentire cyclin A, cyclin E, or cdk2 open reading frame was confirmed bydirect DNA sequencing. pSG5-TETr-cyclin A (E220A) and pSG5-TETr-cyclin E(L134A/Q174A) were generated using Transformer Site-Directed MutagenesisKit (Clontech) according to manufacture's instructions usingpSG5-TETr-cyclin A and pSG5-TETr-cyclin E as a template, respectively,and confirmed by DNA sequencing.

Antibodies and Immunoblot Analysis

Monoclonal anti-TETr was purchased from Clontech and anti-HA (12CA5) waspurchased from Boehringer Mannheim. Polyclonal anti-cyclin A (SC-751),monoclonal and polyclonal anti-cyclin E (SC-247, SC-481), and polyclonalanti-cdk2 (SC-163) were purchased from Santa Cruz. Cell extracts weremade by lysis in EBC buffer (50mM Tris [pH8], 120 mM NaCl, 0.5% NonidetP-40). For immunoblot analysis, ˜100 μg of cell extract was loaded perlane. Nitrocellulose filters were blocked in 4% powdered milk/1% goatserum in TBS-T (10 mM Tris [pH 8], 0.05% Tween, 150 mM NaC1) for 1 hourat room temperature prior to incubation in primary antibody. Anti-HA(12CA5) was used at a concentration of 1.0 μg/ml, anti-TETr antibody at1:500 dilution (v/v), anti-cyclin A (SC-751) at 1:1,000 dilution (v/v),anti-cyclin E (SC-247, SC-481) at 1:1,000 dilution (v/v) and anti-cdk2(SC-163) at 1:1,000 dilution (v/v). Following 4 washes with TBS/T, boundantibody was detected using alkaline phosphatase-conjugated secondaryantibodies.

GST Pull-Down Assay

Glutathione S-transferase pull-down assays were performed basically asdescribed previously (Kaelin et al., 1991). Binding reactions contained10 μl of ³⁵S-radiolabelled in vitro translates made with a TNT kit(Promega) and approximately 1 μg of the indicated GST fusion protein in1 ml of NETN (20mM Tris [pH 8], 100 mM NaCl, 1 mM EDTA, 0.5% NonidetP-40). Following 1 hour incubation at 4° C. with rocking, the Sepharosewas washed 5 times with NETN. Bound proteins were eluted by boiling inSDS-containing sample buffer and resolved by SDS-polyacrylamide gelelectrophoresis. Comparable loading of GST-fusion proteins was confirmedby Coomassie brilliant blue staining and ³⁵S-radiolabelled proteins weredetected by fluorography.

FACS/Cell Cycle Analysis

Fluorescence activated cell sorting (FACS) was done essentially asdescribed (Qin et al., 1995). Briefly, subconfluent SAOS-2 cells grownin 100 mm dishes were transfected with 2 μg of pCD19 and 10 μg ofpSG5-HA-RB together with plasmids encoding the indicated cyclins. 72 hrlater the cells were harvested with trypsin-EDTA and stained withFITC-conjugated anti-CD19 antibody (CALTAG) and propiodium iodide.Samples were analyzed by two-color FACS with a FACScan (BectonDickinson). For cell cycle synchronization, cells were starved inserum-free DMEM for 72 hours before being stimulated with 10% FBS.

Luciferase Reporter Gene Assay

For TETr-fusion transcriptional assay, subconfluent U2OS cells weretransiently transfected in 6-well plates in duplicate with 1 μg ofpCMV-μgal, 1 μg of pUHC13-3 reporter plasmid, and 3 μg of the indicatedplasmids encoding TETr-fusion proteins. Sufficient parental pSG5-TETrwas added so that each reaction contained the same amount of pSG5-TETrbackbone. 48 hours after transfection luciferase activity andβ-galactosidase activity was determined as described previously (Qin,1995).

In Vitro Kinase Assay

500 μg of cell extract was incubated with protein A Sepharose and 1 μgof anti-cyclin E (SC-481) or anti-cyclin A (SC-751) antibody for 1 hourat 4° C. in a final volume of 0.5 mL. The Sepharose was then washed 5times with NETN and 3 times in IP kinase (IPK) buffer (50 mM Tris-HC1[pH 7.5], 10 mM MgCl₂, 1 mM DTT). The Sepharose was then resuspended in27 μl of IPK buffer to which was added 2 μl of histone H1 (1 mg/ml) and1 μl of [γ⁻³²P] ATP (6,000 Ci/mmol, 10 mCi/ml) and incubated for 30 minat 30° C. Reactions were stopped by addition of Laemmli sample buffer,boiled, resolved by SDS-polyacrylamidel gel electrophoresis, andsubjected to autoradiography.

References:

G. Semenza, Annu. Rev. Cell Dev. Biol. 15, 551-578 (1999).

R. Wenger, J Exper Biol. 203, 1253-1263 (2000). G. Semenza, Cell 98,281-4 (1999).

H. Zhu, F. Bunn, Resp. Phys. 115, 239-247 (1999).

M. Ivan, W. G. Kaelin, Current Opinion in Gen and Dev In Press (2000).E. Maher, W. G. Kaelin, Medicine 76, 381-91 (1997). M. Tyers, R.Rottapel, Proc Nall Acad Sci USA 1999 Oct. 26;96(22): 12230-2 96,12230-2 (1999). W. Krek, Nat Cell Biol. 2000 July; 2(7):E121-3 2, E121-3(2000). C. E. Stebbins, W. G. Kaelin, N. P. Pavletich, Science 284,455-461 (1999). M. Ohh, et al., Nature Cell Biology 2 , 423-427 (2000).

T. Kamura, et al., Proc. Natl. Acad. Sci. (USA) 97, 10430-10435 (2000).M. Cockman, et al., J Biol. Chem 275, 25733-41 (2000).

K. Tanimoto, Y. Makino, T. Pereira, L. Poellinger, EMBO J 19, 4298-4309(2000). P. Maxwell, et al., Nature 399, 271-5 (1999). M. A. Goldberg, S.P. Dunning, H. F. Bunn, Science 242, 1412-1415 (1988). G. Wang, G.Semenza, Blood 82, 3610-5 (1993). V. Ho, H. Bunn, Biochem Biophys ResCommun 1996 Jun. 5; 223(1):175-80 223, 175-80 (1996). D. Chowdary, J.Dermody, K. Jha, H. Ozer, Mol Cell Biol 14, 1997-2003 (1994). L. E.Huang, J. Gu, M. Schau, H. F. Bunn, Proc Natl Acad Sci U S A 95, 7987-92(1998).

C. Pugh, 1. O'Rourke, M. Nagao, J. Gleadle, P. Ratcliffe, J Biol. Chem272, 11205-14 (1997).

V. Srinivas, L. Zhang, X. Zhu, J. Caro, Biochem Biophys Res Commun 260,557-61 (1999). K. I. Kivirikko, J. Myllyharju, Matrix Biology 16,357-368 (1998). A. Winter, A. Page, Mol Cell Biol. 20, 4084-4093 (2000).L. Friedman, et al., Proc Natl Acad Sci USA 97, 4736-41 (2000). OIliopoulos, A. Kibel, S. Gray, W. G. Kaelin, Nature Medicine 1, 822-826(1995). R. Deshaies, Annu Rev Cell Dev Biol 15, 435-67 (1999). Y.Takahashi, S. Takahashi, Y. Shiga, T. Yoshimi, T. Miura, J Biol Chem275, 14139-46 (2000). C. Levene, C. Bates, Biochim Biophys Acta 444,446-52 (1976). L. Huang, W. Willmore, J. Gu, M. Goldberg, H. Bunn, JBiol Chem 274, 9038-44 (1999). Y. Liu, et al., J Biol Chem 273, 15257-62(1998). T. Morita, S. Kourembanas, J Clin Invest 96, 2676-82 (1995). C.Sutter, E. Laughner, G. Semenza, Proc Natl Acad Sci USA 97, 4748-53(2000). G. Wang, B. Jiang, G. Semenza, Biochem Biophys Res Commun 1995Nov. 13; 216(2):669-75 216, 669-75 (1995). K. Sogawa, et al., Proc NatlAcad Sci U S A 1998 Jun. 23; 95(13):7368-73 95,7368-73 (1998). S.Salceda, I. Beck, V. Srinivas, J. Caro, Kidney Int 1997 February;51(2):556-9 51, 556-9 (1997). J. Wingrove, P. O'Farrell, Cell 1999 Jul9; 98(1):105-14 98, 105-14 (1999). L. Palmer, G. Semenza, M. Stoler, R.Johns, Am J Physiol 274, L212-9 (1998). G. Melillo, et al., J Exp Med1995 Dec. 1; 182(6):1683-93 182,1683-93 (1995). M. Bickel, et al.,Hepatology 28, 404-11 (1998). T. Franklin, W. Morris, P. Edwards, M.Large, R. Stephenson, Biochem J 353, 333-338 (2001).

Adams, P. D., Sellers, W. R., Sharma, S. K., Wu, A. D., Nalin, C. M. andKaelin, W. G. (1996) Identification of a Cyclin-cdk2 recognition motifpresent in substrates and p21-like cdk inhibitors. Mol Cell. Biol. 16,6623-6633Adams, P. D., Li, X., Sellers, W. R., Baker, K. B., Leng, X., Harper, J.W., Taya, Y. and Kaelin, W. G. (1999) The retinoblastoma proteincontains a C-terminal motif that targets it for phosphorylation bycyclin/cdk complexes. Mol. Cell.Biol. 19, 1068-1080Akoulitchev, S., Chuikov, S., Reinberg, D. (2000) TFIIH is negativelyregulated by cdk8-containing mediator complexes. Nature, 407, 102-6Bagby, S., Kim, S., Maldonado, E., Tong, K. I., Reinberg, D. and Ikura,M. (1995) Solution structure of the C-terminal core domain of humanTFIIB : similarity to cyclin A and interaction with TATA-bindingprotein. Cell 82, 857-867Bandara, L. R., Adamczewski, J. P., Hunt, T. and La Thangue, N. B.(1991) Cyclin A and the retinoblastoma gene product complex with acommon transcription factor. Nature 352, 249-251Beijersbergen, R. L., Carlee, L., Kerkhoven, R. M. and Bernards, R.(1995) Regulation of the retinoblastoma protein-related p107 by G1cyclin complexes. Genes Dev. 9, 1340-1353Bieniasz, P. D., Grdina, T. A., Bogerd, H. P. and Cullen, B. R. (1999)Recruitment of cyclin T1/P-TEFb to an HIV type 1 long terminal repeatpromoter proximal RNA target is both necessary and sufficient for fullactivation of transcription. Proc. Natl. Acad. Sci. USA 96, 7791-7796Bochar, D. A., Pan, Z. Q., Knights, R., Fisher, R. P., Shilatifard, A.and Shiekhattar, R. (1999) Inhibition of transcription by the trimericcyclin-dependent kinase 7 complex. Biol. Chem. 274, 13162-13166Chen, C. and Okayama, H. (1987) High-efficiency transformation ofmammalian cells by plasmid DNA. Mol. Cell. Biol. 7, 2745-2752Chen, J., Saha, P., Kornbluth, S., Dynlacht, B. D. and Dutta, A. (1996)Cyclin-binding motifs are essential for the function of p21 CIP1. Mol.Cell. Biol. 16, 4673-4682Dahmus, M. E. (1996) Reversible phosphorylation of the C-terminal domainof RNA polymerase II. J Biol. Chem. 271, 19009-19012Devoto, S. H., Mudryj, M., Pines, J., Hunter, T. and Nevins, J. R.(1992) A cyclin A protein kinase complex possesses sequence specific DNAbinding activity; p33cdk2 is a component of the E2F-Cyclin A complex.Cell 68, 167-176Dynlacht, B. D. (1997) Regulation of transcription by proteins thatcontrol the cell cycle. Nature 389, 149-152Dynlacht, B. D., Brook, A., Dembski, M., Yenush, L. and Dyson, N. (1994)DNA-binding and trans-activation properties of drosophila E2F and DPproteins. Proc. Natl. Acad. Sci. USA 91, 6359-6363Dynlacht, B. D., Moberg, K., Lees, J. A., Harlow, E. and Zhu, L. (1997)Specific regulation of E2F family members by cyclin-dependent kinases.Mol. Cell. Biol. 17, 3867-3875Ewen, M. E., Faha, B., Harlow, E. and Livingston, D. M. (1992)Interaction of p107 with cyclin A independent of complex formation withviral oncoproteins. Science 255, 85-87Faha, B., Ewen, M., Tsai, L., Livingston, D. M. and Harlow, E. (1992)Interaction between human cyclin A and adenovirus E1A-associated p107Protein. Science 255, 87-90Felzen, L. K., Farrell, S., Betts, J. C., Mosavin, R. and Nabel, G. J.(1999) Specificity of cyclin-cdk2, TFIIB, and E1A interactions with acommon domain of the p300 coactivator. Mol. Cell. Biol. 19, 4241-4246Fu, T. J., Peng, J., Lee, G., Price, D. H. and Flores, 0. (1999) CyclinK functions as a CDK9 regulatory subunit and participates in RNApolymerase II transcription. J Biol. Chem. 274, 34527-34530Gebara, M. M., Sayre, M. H. and Corden, J. L. (1997) Phosphorylation ofthe carboxy-terminal repeat domain in RNA polymerase II bycyclin-dependent kinases is sufficient to inhibit transcription. J Cell.Biochem. 64, 390-402Gossen, M. and Bujard, H. (1992) Tight control of gene expression inmammalian cells by tetracycline-responsive promoters. Proc. Natl. Acad.Sci. USA 89, 5547-5551Hannon, G. J., Demetrick, D. and Beach, D. (1993) Isolation of theRb-related p130 through its interactions with CDK2 and cyclins. GenesDev. 7, 2378-2391Hengartner, C. J., Myer, V. E., Liao, S. M., J., W. C., Koh, S. S. andYoung, R. A. (1998) Temporal regulation of RNA polymerase II by Srb10and Kin28 cyclin-dependent kinases. Mol. Cell 2, 43-53Jackson, P. K., Chevalier, S., Philippe, M. and Kirschner, M. W. (1995)Early events in DNA replication require cyclin E and are blocked byp21CIP1. J Cell. Biol. 130, 755-69Jeffrey, P., Gonna, S. and Pavletich, N. (1995) Crystal structure of thetetramerization domain of the p53 tumor suppressor at 1.7 angstroms.Science 267, 1498-1502Jones, K. A. (1997) Taking a new TAK on Tat transactivation. Genes Dev.11, 2593-2599Kaelin, W. G., Pallas, D. C., DeCaprio, J. A., Kaye, F. J. andLivingston, D. M. (1991) Identification of cellular proteins that caninteract specifically with the T/E1A-binding region of theretinoblastoma gene product. Cell 64, 521-532Kimmelman, J., Kaldis, P., Hengartner, C. J., Laff, G. M., Koh, S. S.,Young, R. A. and Solomon, M. J. (1999) Activating phosphorylation of theKin28p subunit of yeast TFIIH by Cak1p. Mol. Cell. Biol. 19, 4774-4787Krek, W., Ewen, M., Shirodkar, S., Arany, Z., Kaelin, W. G. andLivingston, D. M. (1994) Negative regulation of the growth-promotingtranscription Factor E2F-1 by a stably bound cyclin A-dependent proteinkinase. Cell 78, 1-20Lania, L., Majello, B. and Napolitano, G. (1999) Transcriptional controlby cell-cycle regulators: a review. J Cell. Physiol. 179, 134-141Lee, M. H., Williams, B. O., Mulligan, G., Mukai, S., Bronson, R. T.,Dyson, N., Harlow, E. and Jacks, T. (1996) Targeted disruption of p107:functional overlap between p107 and Rb. Genes Dev. 10, 1621-1632Lees, E., Faha, B., Dulic, V., Reed, S. I. and Harlow, E. (1992) CyclinE/cdk2 and cyclin A/cdk2 kinases associate with p107 and E2F in atemporally distinct manner. Genes Dev. 6, 1874-1885Leresche, A., Wolf, V. J. and Gottesfeld, J. M. (1996) Repression of RNApolymerase II and III transcription during M phase of the cell cycle.Exp Cell Res. 229, 282-288Ma, T., Van Tine, B. A., Wei, Y, Garrett, M. D., Nelson, D., Adams, P.D., Wang, J., Qin, J., Chow, L. T., Harper, J. W. (2000) Cellcycle-regulated phosphorylation of p220NPAT by cyclin E/Cdk2 in Cajalbodies promotes histone gene transcription. Genes Dev., 14, 2298-2313Majello, B., Napolitano, G., Giordano, A. and Lania, L. (1999)Transcriptional regulation by targeted recruitment of cyclin-dependentCDK9 kinase in vivo. Oncogene, 18, 4598-4605Mudryj, M., Devoto, S. H., Hiebert, S. W., Hunter, T., Pines, J. andNevins, J. R. (1991) Cell cycle regulation of the E2F transcriptionfactor involves an interactin with cyclin A. Cell 65, 1243-1253Neuman, E., Ladha, M. H., Lin, N., Upton, T. M., Miller, S. J., DiRenzo,J., Pestell, R. G., Hinds, P. W., Dowdy, S. F., Brown, M., Ewen, M.E.(1997) Cyclin D1 stimulation of estrogen receptor transcriptionalactivity independent of cdk4. Mol Cell Biol 17, 5338-47Noble, M. E., Endicott, J. A., Brown, N. R. and Johnson, L. N. (1997)The cyclin box fold: protein recognition in cell-cycle and transcriptioncontrol. Trends Biochem Sci. 22, 482-487Peeper, D. S., Parker, L. L., Ewen, M. E., Toebes, M., Frederick, F. L.,Xu, M., Zantema, A., van der Eb, A. J. and Pinwica-Worms, H. (1993) A-and B- type cyclins differentially modulate substrate specificity ofcyclin-CDK complexes. EMBO J 112, 1947-1954Perkins, N. D., Felzen, L. K., Betts, J. C., Leung, K., Beach, D. H. andN abel, G. J. (1997) Regulation of NF-kappaB by cyclin-dependent Kinasesassociated with the p300 Coactivator. Science 275, 523-527Qin, X.-Q., Livingston, D. M., Ewen, M., Sellers, W. R., Arany, Z., andKaelin, W. G. (1995) The transcription factor E2F1 is a downstreamtarget of RB action. Mol. Biol. Cell. 15, 742-755Rickert, P., Corden, J. L. and Lees, E. (1999) Cyelin C/CDK8 and cyclinH/CDK7/p36 are biochemically distinct CTD kinases. Oncogene 18,1093-1102Roberts, J. M. (1999) Evolving ideas about cyclins. Cell 98, 129-132Schulman, B., Lindstrom, D. and Harlow, E.: (1998) Substrate recruitmentto cyclin-dependent kinase 2 by a multipurpose docking site on cyclin A.Proc. Natl. Acad. Sci. USA 95, 10453-10458Schwarz, J. K., Devoto, S. H., Smith, E. J., Chellappan, S. P., Jakoi,L. and Nevins, J. R. (1993) Interactions of the p107 and Rb proteinswith E2F during the cell proliferation response. EMBO J 1 12, 1013-1020Sellers, W. R., Neuman, E. and Kaelin, W. G. (1995) The Retinoblastomaprotein contains a potent transrepression domain which Induces acell-cycle block when bound to DNA. Proc. Natl. Acad. Sci. USA 92,11544-11548Shanahan, F., Seghezzi, W., Parry, D., Mahony, D. and Lees, E. (1999)Cyclin E associates with BAF155 and BRG1, components of the mammalianSWI-SNF complex, and alters the ability of BRG1 to induce growth arrest.Mol. Cell. Biol. 19, 1460-1469Sherr, C J. (1996) Cancer Cell Cycles. Science, 274, 1672-1677Smith, E. J., Leone, G. and Nevins, J. R. (1998) Distinct mechanismscontrol the accumulation of the Rb-related p107 and p130 proteins duringcell growth. Cell Growth Differ. 9, 297-303Starostik, P., Chow, K. N, and Dean, D. C. (1996) Transcriptionalrepression and growth suppression by the p107 pocket protein. Mol. Cell.Biol. 16, 3606-3614Tedder, T. F. and Isaacs, C. M. (1989) Isolation of cDNAs Encoding theCD19 Antigen of Human and Mouse B Lymphocytes. J. Immunology 143,712-717van den Heuvel, S. and Harlow, E. (1993) Distinct roles forcyclin-dependent kinases in cell cycle control. Science 262, 2050-2053Weinberg, R. A. (1995) The retinoblastoma protein and cell cyclecontrol. Cell 81, 323-330Xu, M., Sheppard, K. A., Peng, C-Y., Yee, A. S., and Piwnica-Worms, H.(1994) Cyclin A/cdk2 binds directly to E2F1 and inhibits the DNA-bindingactivity of E2F1/DP1 by phosphorylation. Mol. Cell. Biol. 14, 8420-8431Yankulov, K. Y. and Bentley, D. L. (1997) Regulation of CDK7 substratespecificity by MAT1 and TFIIH. EMBO J. 16, 1638-1646Zamanian, M. and La Thangue, N. B. (1993) Transcriptional repression bythe RB-related protein p107. Mol. Biol. Cell 4, 389-396Zhao, J., Dynlacht, B., lmai, T., Hon, T. and Harlow, E. (1998)Expression of NPAT, a novel substrate of cyclin E-CDK2, promotes S-phaseentry. Genes Dev. 12, 456-461Zhao, J., Kennedy, B. K., Lawrence, B. D., Barbie, D. A., Matera, A. G.,Fletcher, J. A. and Harlow, E. (2000) NPAT links cyclin E-Cdk2 to theregulation of replication-dependent histone gene transcription. Genesand Dev. 14, 2283-2297Zhu, L., Enders, G., Lees, J. A., Beijersbergen, R. L., Bernards, R. andHarlow, E. (1995a) The pRB-related protein p107 contains two growthsuppression domains: Independent interactions with E2F and cyclin/cdkcomplexes. EMBO J. 14, 1904-1913Zhu, L., Harlow, E. and Dynlacht, B. D. (1995b) p107 uses ap21/CIP1-related domain to bind cyclin/cdk2 and regulate interactionswith E2F. Genes Dev. 9, 1740-1752Zhu, Y., Pe'ery, T., Peng, J., Ramanathan, Y., Marshall, N., Marshall,T., Amendt, B., Mathews, M.B. and Price, D.H. (1997) Transcriptionelongation factor P-TEFb is required for HIV-1 tat transactivation invitro. Genes Dev. 11, 2622-2632Zwijsen, R. M., Wientjens, E., Klompmaker, R., van der Sman, J.,Bernards, R., Michalides, R J. (1997) CDK-independent activation ofestrogen receptor by cyclin D1. Cell 88, 405-15Zwijsen, R. M., Buckle, R. S., Hijmans, E. M., Loomans, C. J., Bernards,R. (1998) Ligand-independent recruitment of steroid receptorcoactivators to estrogen receptor by cyclin D1. Genes Dev. 12, 3488-98

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents are considered tobe within the scope of the present invention and are covered by thefollowing claims. The contents of all references, issued patents, andpublished patent applications cited throughout this application arehereby incorporated by reference. The appropriate components, processes,and methods of those patents, applications and other documents may beselected for the present invention and embodiments thereof.

1-14. (canceled)
 15. A method of treating or preventing a hypoxic orischemic related disorder in a subject, comprising administering to asubject in need thereof a compound which decreases prolyl hydroxylaseexpression or activity, such that said hypoxic or ischemic relateddisorder is treated, wherein the compound is identified by an assaycomprising the steps of: a) bringing into contact a prolyl hydroxylase,a HIF1α polypeptide moiety consisting of amino acids 555-575 of wildtype HIFα, numbered in accordance with wild type HIFα, and a putativemodulator compound under conditions where prolyl hydroxylase, in theabsence of modulator, is capable of acting on said HIF1α polypeptidemoiety; and b) measuring the degree of prolyl hydroxylase inhibitioncaused by said modulator compound. 16-21. (canceled)
 22. A method ofincreasing angiogenesis or vascularization in a subject, comprisingadministering to a subject in need thereof a compound which decreasesprolyl hydroxylase expression or activity identified by an assaycomprising the steps of: a) bringing into contact a prolyl hydroxylase,a HIF1α polypeptide moiety consisting of amino acids 555-575 of wildtype HIFα, numbered in accordance with wild type HIFα, and a putativemodulator compound under conditions where prolyl hydroxylase, in theabsence of modulator, is capable of acting on said HIF1α polypeptidemoiety; and b) measuring the degree of prolyl hydroxylase inhibitioncaused by said modulator compound. 23-54. (canceled)
 55. The method ofclaim 15, wherein said assay further comprises pVHL.
 56. The method ofclaim 15, wherein the hypoxic or ischemic related disorder is tissueischemia.
 57. The method of claim 22, wherein said assay furthercomprises pVHL.